Il-1 binding proteins

ABSTRACT

Proteins that bind IL-1α and IL-1β are described along with their use in compositions and methods for treating, preventing, and diagnosing IL-1-related disorders and for detecting IL-1α and IL-1β in cells, tissues, samples, and compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 61/334,917, filed May 14, 2010, and of U.S. ProvisionalApplication No. 61/425,701, filed Dec. 21, 2010, the contents of whichare incorporated herein in their entireties by reference.

FIELD OF THE INVENTION

The present invention relates to IL-1 binding proteins, and specificallyto their uses in the prevention and/or treatment of acute and chronicimmunological diseases such as rheumatoid arthritis, osteoarthritis,psoriasis, multiple sclerosis, and other autoimmune diseases.

BACKGROUND OF THE INVENTION

Cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF),are molecules produced by a variety of cells, such as monocytes andmacrophages, that are mediators of inflammatory processes. Interleukin-1is a cytokine with a wide range of biological and physiological effects,including fever, prostaglandin synthesis (m, e.g., fibroblasts, musclecells and endothelial cells), T-lymphocyte activation, and interleukin-2production.

The original members of the IL-1 superfamily are IL-1α, IL-1β, and theIL-1 Receptor antagonist (IL-1Ra, IL-1RA, IL-1ra, IL-1Rα). IL-1α andIL-1β are pro-inflammatory cytokines involved in immune defense againstinfection. The IL-1Rα is a molecule that competes for receptor bindingwith IL-1α and IL-1β, blocking their role in immune activation. Recentyears have seen the addition of other molecules to the IL-1 superfamilyincluding IL-18 (see Dinarello et al., FASEB J., 8(15):1314-3225 (1994);Huising et al., Dev. Comp. Immunol., 28(5):395-413 (2004)) and six moregenes with structural homology to IL-1α, IL-1β, or IL-1RA. These lattersix members are named IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, and IL1F10. Inaccordance, IL-1α, IL-1β, and IL-1RA have been renamed IL-1F1, IL-1F2,and IL-1F3, respectively (see Sims et al., Trends Immunol., 22(10):536-537 (2001); Dunn et al., Trends Immunol., 22(10): 533-536 (2001)). Afurther putative member of the IL-1 family has been described calledIL-33 or IL-1F11, although this name is not officially accepted in theHGNC gene family nomenclature database.

Both IL-1α and IL-1β are produced by macrophages, monocytes anddendritic cells. They form an important part of the inflammatoryresponse of the body against infection. These cytokines increase theexpression of adhesion factors on endothelial cells to enabletransmigration of leukocytes, the cells that fight pathogens, to sitesof infection and re-set the hypothalamus thermoregulatory center,leading to an increased body temperature which expresses itself asfever. IL-1 is therefore called an endogenous pyrogen. The increasedbody temperature helps the body's immune system to fight infection. IL-1is also important in the regulation of hematopoiesis. IL-1β productionin peripheral tissue has also been associated with hyperalgesia(increased sensitivity to pain) associated with fever (Morgan et al.,Brain Res., 1022(1-2): 96-100 (2004)). For the most part, these twoforms of IL-1 bind to the same cellular receptor. This receptor iscomposed of two related, but non-identical, subunits that transmitintracellular signals via a pathway that is mostly shared with certainother receptors. These include the Toll family of innate immunereceptors and the receptor for IL-18. IL-1α and IL-1β also possesssimilar biological properties, including induction of fever, slow wavesleep, and neutrophilia, T- and B-lymphocyte activation, fibroblastproliferation, cytotoxicity for certain cells, induction ofcollagenases, synthesis of hepatic acute phase proteins, and increasedproduction of colony stimulating factors and collagen.

cDNAs encoding the two distinct forms of IL-1 have been isolated andexpressed; these cDNAs represent two different gene products, termedIL-1β (Auron et al., Proc. Natl. Acad. Sci. USA, 81: 7907-7911 (1984))and IL-1α (Lomedico et al., Nature, 312: 458-462 (1984)). IL-1β is thepredominant form produced by human monocytes both at the mRNA andprotein levels. The two forms of human IL-1 share only 26% amino acidhomology. Despite their distinct polypeptide sequences, the two forms ofIL-1 have structural similarities (Auron et al., J. Mol. Cell. Immunol.,2: 169-177 (1985)), in that the amino acid homology is confined todiscrete regions of the IL-1 molecule.

IL-1α and IL-1β are produced as precursor peptides. In other words theyare made as a long protein that is then processed to release a shorter,active molecule, which is called the mature protein. Mature IL-1β, forexample, is released from Pro-IL-1β following cleavage by a certainmember of the caspase family of proteins, called caspase-1 or theinterleukin-1 converting enzyme (ICE). The 3-dimensional structure ofthe mature forms of each member of the human IL-1 superfamily iscomposed of 12-14 β-strands producing a barrel-shaped protein.

Although a variety of antibodies to IL-1 have been described in thenearly two decades of work since the discovery of this criticalproinflammatory cytokine, there remains a need for improved antibodiesthat can effectively mediate or neutralize the activity of IL-1 in theinflammatory response and autoimmune disorders and for use in detectingIL-1β in samples and tissues.

SUMMARY OF THE INVENTION

This invention pertains to proteins that bind human IL-1α and IL-1β.Binding proteins of the invention include but are not limited toantibodies, antigen binding portions thereof, and multivalent,multispecific binding proteins such as DVD-Ig™ binding proteins that canbind human IL-1α and IL-1β. The invention also provides methods ofmaking and using the IL-1α and IL-1β binding proteins described hereinas well as various compositions that may be used in methods of detectingIL-1α and IL-1β in a sample or in methods of treating or preventing adisorder in an individual that is associated with or suspected to beassociated with IL-1 activity.

In an embodiment, the invention provides a binding protein comprisingfirst and second polypeptide chains, wherein said first polypeptidechain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein:

-   -   VD1 is a first heavy chain variable domain;    -   VD2 is a second heavy chain variable domain;    -   C is a heavy chain constant domain;    -   X1 is a linker with the proviso that it is not CH1;    -   X2 is an Fc region; and    -   n is independently 0 or 1; and    -   wherein said second polypeptide chain comprises a second        VD1-(X1)n-VD2-C-(X2)n, wherein:    -   VD1 is a first light chain variable domain;    -   VD2 is a second light chain variable domain;    -   C is a light chain constant domain;    -   X1 is a linker with the proviso that it is not CH1;    -   X2 does not comprise an Fc region; and    -   n is independently 0 or 1;

wherein, in said first polypeptide chain, VD1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 60-148, 196,198, 200, 202, 204, 206, 208 and 210; and VD2 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 213 and 227;

wherein, in said second polypeptide chain, VD1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 149-189, 197,199, 201, 203, 205, 207, 209 and 211; and VD2 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 216 and 229;and

wherein the binding protein binds human IL-1β and human IL-1α.

In an embodiment, a binding protein described above comprises a firstpolypeptide chain that comprises an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 212, 217, 226, 230, 232, 234, and236.

In another embodiment, a binding protein described above comprises asecond polypeptide chain that comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 215, 218, 228, 231, 233, 235,and 237.

In another aspect of the invention, a binding protein comprises firstand second polypeptide chains, wherein said first polypeptide chaincomprises a first VD1-(X1)n-VD2-C-(X2)n, wherein:

-   -   VD1 is a first heavy chain variable domain;    -   VD2 is a second heavy chain variable domain;    -   C is a heavy chain constant domain;    -   X1 is a linker with the proviso that it is not CH1;    -   X2 is an Fc region; and    -   n is independently 0 or 1; and    -   wherein said second polypeptide chain comprises a second        VD1-(X1)n-VD2-C-(X2)n, wherein:    -   VD1 is a first light chain variable domain;    -   VD2 is a second light chain variable domain;    -   C is a light chain constant domain;    -   X1 is a linker with the proviso that it is not CH1;    -   X2 does not comprise an Fc region; and    -   n is independently 0 or 1;

wherein, in said first polypeptide chain, VD1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 213 and 227;and VD2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 60-148, 196, 198, 200, 202, 204, 206, 208 and210;

wherein, in said second polypeptide chain, VD1 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 216 and 229;and VD2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 149-189, 197, 199, 201, 203, 205, 207, 209 and211; and wherein the binding protein binds human IL-1β and human IL-1α.

In another embodiment, a binding protein described above comprises afirst polypeptide chain that comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 219 and 221.

In another embodiment, a binding protein described above comprises asecond polypeptide chain that comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 220 and 222.

In another aspect, the invention provides a binding protein describedabove, wherein:

-   -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:212, then said second polypeptide chain        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 215, 228, 231, 233 and 235;    -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:217, then said second polypeptide chain        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 218 and 237;    -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:219, then said second polypeptide chain        comprises the amino acid sequence of SEQ ID NO:220;    -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:221, then said second polypeptide chain        comprises SEQ ID NO:222;    -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:226, then said second polypeptide chain        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 228, 215, 231, 233, and 235;    -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:230, then said second polypeptide chain        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 231, 215, 228, 233, and 235;    -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:232, then said second polypeptide chain        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 233, 215, 228, 231, and 235;    -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:234, then said second polypeptide chain        comprises an amino acid sequence selected from the group        consisting of SEQ ID NOs: 235, 215, 228, 231, and 233; and    -   when said first polypeptide chain comprises the amino acid        sequence of SEQ ID NO:236, then said second polypeptide chain        comprises SEQ ID NO:237.

In another aspect, the invention provides a protein described above,wherein:

-   -   said first polypeptide chain comprises SEQ ID NO:212, and said        second polypeptide chain comprises SEQ ID NO:215; or    -   said first polypeptide chain comprises SEQ ID NO:217, and said        second polypeptide chain comprises SEQ ID NO:218; or    -   said first polypeptide chain comprises SEQ ID NO:219, and said        second polypeptide chain comprises SEQ ID NO:220; or    -   said first polypeptide chain comprises SEQ ID NO:221, and said        second polypeptide chain comprises SEQ ID NO:222; or    -   said first polypeptide chain comprises SEQ ID NO:226, and said        second polypeptide chain comprises SEQ ID NO:228; or    -   said first polypeptide chain comprises SEQ ID NO:230, and said        second polypeptide chain comprises SEQ ID NO:231; or    -   said first polypeptide chain comprises SEQ ID NO:232, and said        second polypeptide chain comprises SEQ ID NO:233; or    -   said first polypeptide chain comprises SEQ ID NO:234, and said        second polypeptide chain comprises SEQ ID NO:235; or    -   said first polypeptide chain comprises SEQ ID NO:236, and said        second polypeptide chain comprises SEQ ID NO:237.

In an embodiment, a binding protein of the invention described abovecomprises two first polypeptide chains and two second polypeptidechains.

In another aspect, in a binding protein described above X1 or X2 is anamino acid sequence selected from the group consisting of SEQ IDNOs:26-57, 233, 224, and 225.

In another embodiment, the invention provides a binding proteindescribed above wherein the Fc region is selected from the groupconsisting of a native sequence Fc region and a variant sequence Fcregion. In another embodiment, the Fc region is selected from the groupconsisting of an Fc region from an IgG1, IgG2, IgG3, IgG4, IgA, IgM,IgE, and IgD.

In another embodiment, the invention provides a binding proteinconjugate that comprises a binding protein described above and furthercomprises an agent. Such agents include, but are not limited to, animmunoadhesion molecule, an imaging agent, a therapeutic agent, and acytotoxic agent. Preferred imaging agents include, but are not limitedto, a radiolabel, an enzyme, a fluorescent label, a luminescent label, abioluminescent label, a magnetic label, and biotin. Preferredradiolabels include, but are not limited to, ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. A preferred therapeutic orcytotoxic agent includes, but is not limited to, an anti-metabolite, analkylating agent, an antibiotic, a growth factor, a cytokine, ananti-angiogenic agent, an anti-mitotic agent, an anthracycline, toxin,and an apoptotic agent.

The invention also provides a binding protein comprising an antigenbinding domain, wherein the binding protein is capable of binding humanIL-1β and the antigen-binding domain comprises six CDRs, i.e., CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, as defined below:

-   -   CDR-H1: X1-Y-D-M-S (SEQ ID NO:190), wherein;        -   X₁ is S, K or R;    -   CDR-H2: Y-X₂-S-X₄-G-G-X₇-G-T-Y-Y-P-D-X₁₄-X₁₅-K-G (SEQ ID NO:        191),        -   wherein;        -   X₂ is I or V;        -   X₄ is S or H;        -   X₇ is G or A;        -   X₁₄ is T or S; and        -   X₁₅ is V or A;    -   CDR-H3: G-G-V-X₄-K-G-X₇-F-D-X10 (SEQ ID NO:192), wherein;        -   X₄ is T or Y;        -   X₇ is Y or C; and        -   X₁₀ is V, E, L, M, Q, or Y;    -   CDR-L1: R-A-S-G-N-I-X₇-X₈-X₉-L-X₁₁ (SEQ ID NO:193), wherein;        -   X₇ is H, Y, or W;        -   X₈ is N, G, T, Q, E, H, D, or K;        -   X₉ is Y or W; and        -   X₁₁ is T, A, or N;    -   CDR-L2: X₁-A-K-X₄-L-X₆-X₇ (SEQ ID NO:194), wherein;        -   X₁ is N, Q, or D;        -   X₄ is T, N, I, E, or S;        -   X₆ is A, M, or E; and        -   X₇ is D, E, 5, or A;    -   and    -   CDR-L3: Q-X₂-F-W-X₅-X₆-P-X₈-X₉ (SEQ ID NO: 195), wherein;        -   X₂ is H or Q;        -   X₅ is S, N, T, K, R, or M;        -   X₆ is I or L;        -   X₈ is Y or A; and        -   X₉ is T, I, and N;    -   except that when CDR-H1 is S-Y-D-M-S (SEQ ID NO:17), then:    -   CDR-H2 cannot be Y-1-S-S-G-G-G-G-T-Y-Y-F-D-T-V-K-G (SEQ ID        NO:18);    -   CDR-H3 cannot be G-G-V-T-K-G-Y-F-D-V (SEQ ID NO: 19);    -   CDR-L1 cannot be R-A-S-G-N-I-H-N-Y-L-T (SEQ ID NO:20);    -   CDR-L2 cannot be N-A-K-T-L-A-D (SEQ ID NO: 21); and    -   CDR-L3 cannot be Q-H-F-W-S-I-P-Y-T (SEQ ID NO: 22).

In an embodiment, an isolated binding protein described above comprisesat least one CDR that comprises an amino acid sequence selected from thegroup of CDR sequences consisting of:

residues 31-35 of residues 31-35 of SEQ ID NO: 60 SEQ ID NO: 63(CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 of SEQ ID NO: 60SEQ ID NO: 63 (CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 ofSEQ ID NO: 60 SEQ ID NO: 63 (CDR-H3); (CDR-H3); residues 24-34 ofresidues 24-34 of SEQ ID NO: 149 SEQ ID NO: 150 (CDR-L1); (CDR-L1);residues 50-56 of residues 50-56 of SEQ ID NO: 149 SEQ ID NO: 150(CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 of SEQ ID NO: 149SEQ ID NO: 150 (CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 ofSEQ ID NO: 69 SEQ ID NO: 97 (CDR-H1); (CDR-H1); residues 50-66 ofresidues 50-66 of SEQ ID NO: 69 SEQ ID NO: 97 (CDR-H2); (CDR-H2);residues 99-108 of residues 99-108 of SEQ ID NO: 69 SEQ ID NO: 97(CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 of SEQ ID NO: 173SEQ ID NO: 187 (CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 ofSEQ ID NO: 173 SEQ ID NO: 187 (CDR-L2); (CDR-L2); residues 89-97 ofresidues 89-97 of SEQ ID NO: 173 SEQ ID NO: 187 (CDR-L3); (CDR-L3);residues 31-35 of residues 31-35 of SEQ ID NO: 67 SEQ ID NO: 90(CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 of SEQ ID NO: 67SEQ ID NO: 90 (CDR-H2) (CDR-H2) residues 99-108 of residues 99-108 ofSEQ ID NO: 67 SEQ ID NO: 90 (CDR-H3); (CDR-H3); residues 24-34 ofresidues 24-34 of SEQ ID NO: 151 SEQ ID NO: 156 (CDR-L1); (CDR-L1);residues 50-56 of residues 50-56 of SEQ ID NO: 151 SEQ ID NO: 156(CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 of SEQ ID NO: 151SEQ ID NO: 156 (CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 ofSEQ ID NO: 88 SEQ ID NO: 92 (CDR-H1); (CDR-H1); residues 50-66 ofresidues 50-66 of SEQ ID NO: 88 SEQ ID NO: 92 (CDR-H2); (CDR-H2);residues 99-108 of residues 99-108 of SEQ ID NO: 88 SEQ ID NO: 92(CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 of SEQ ID NO: 159SEQ ID NO: 153 (CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 ofSEQ ID NO: 159 SEQ ID NO: 153 (CDR-L2); (CDR-L2); residues 89-97 ofresidues 89-97 of SEQ ID NO: 159 SEQ ID NO: 153 (CDR-L3); (CDR-L3);residues 31-35 of residues 31-35 of SEQ ID NO: 96 SEQ ID NO: 94(CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 of SEQ ID NO: 96SEQ ID NO: 94 (CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 ofSEQ ID NO: 96 SEQ ID NO: 94 (CDR-H3); (CDR-H3); residues 24-34 ofresidues 24-34 of SEQ ID NO: 155 SEQ ID NO: 166 (CDR-L1); (CDR-L1);residues 50-56 of residues 50-56 of SEQ ID NO: 155 SEQ ID NO: 166(CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 of SEQ ID NO: 155SEQ ID NO: 166 (CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 ofSEQ ID NO: 90 SEQ ID NO: 82 (CDR-H1); (CDR-H1); residues 50-66 ofresidues 50-66 of SEQ ID NO: 90 SEQ ID NO: 82 (CDR-H2) (CDR-H2) residues99-108 of residues 99-108 of SEQ ID NO: 90 SEQ ID NO: 82 (CDR-H3);(CDR-H3); residues 24-34 of residues 24-34 of SEQ ID NO: 182 SEQ ID NO:159 (CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO:182 SEQ ID NO: 159 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97of SEQ ID NO: 182 SEQ ID NO: 159 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 90 SEQ ID NO: 101 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 90 SEQ ID NO: 101(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 90SEQ ID NO: 101 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 167 SEQ ID NO: 158 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 167 SEQ ID NO: 158 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 167 SEQ ID NO: 158(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 90SEQ ID NO: 96 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 90 SEQ ID NO: 96 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 90 SEQ ID NO: 96 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 164 SEQ ID NO: 157(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 164SEQ ID NO: 157 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 164 SEQ ID NO: 157 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 73 SEQ ID NO: 70 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 73 SEQ ID NO: 70(CDR-H2); (CDR-H2) residues 99-108 of residues 99-108 of SEQ ID NO: 73SEQ ID NO: 70 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 174 SEQ ID NO: 161 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 174 SEQ ID NO: 161 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 174 SEQ ID NO: 161(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 86SEQ ID NO: 83 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 86 SEQ ID NO: 83 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 86 SEQ ID NO: 83 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 159 SEQ ID NO: 158(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 159SEQ ID NO: 158 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 159 SEQ ID NO: 158 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 90 SEQ ID NO: 102 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 90 SEQ ID NO: 102(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 90SEQ ID NO: 102 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 169 SEQ ID NO: 185 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 169 SEQ ID NO: 185 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 169 SEQ ID NO: 185(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 91SEQ ID NO: 98 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 91 SEQ ID NO: 98 (CDR-H2) (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 91 SEQ ID NO: 98 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 165 SEQ ID NO: 160(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 165SEQ ID NO: 160 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 165 SEQ ID NO: 160 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 76 SEQ ID NO: 74 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 76 SEQ ID NO: 74(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 76SEQ ID NO: 74 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 175 SEQ ID NO: 186 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 175 SEQ ID NO: 186 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 175 SEQ ID NO: 186(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 99SEQ ID NO: 95 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 99 SEQ ID NO: 95 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 99 SEQ ID NO: 95 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 189 SEQ ID NO: 157(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 189SEQ ID NO: 157 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 189 SEQ ID NO: 157 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 100 SEQ ID NO: 72 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 100 SEQ ID NO: 72(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 100SEQ ID NO: 72 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 168 SEQ ID NO: 178 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 168 SEQ ID NO: 178 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 168 SEQ ID NO: 178(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 71SEQ ID NO: 96 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 71 SEQ ID NO: 96 (CDR-H2) (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 71 SEQ ID NO: 96 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 170 SEQ ID NO: 163(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 170SEQ ID NO: 163 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 170 SEQ ID NO: 163 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 84 SEQ ID NO: 77 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 84 SEQ ID NO: 77(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 84SEQ ID NO: 77 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 188 SEQ ID NO: 183 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 188 SEQ ID NO: 183 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 188 SEQ ID NO: 183(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 78SEQ ID NO: 90 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 78 SEQ ID NO: 90 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 78 SEQ ID NO: 90 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 179 SEQ ID NO: 171(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 179SEQ ID NO: 171 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 179 SEQ ID NO: 171 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 93 SEQ ID NO: 79 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 93 SEQ ID NO: 79(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 93SEQ ID NO: 79 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 176 SEQ ID NO: 180 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 176 SEQ ID NO: 180 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 176 SEQ ID NO: 180(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 87SEQ ID NO: 381 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 87 SEQ ID NO: 381 (CDR-H2) (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 87 SEQ ID NO: 381 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 154 SEQ ID NO: 152(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 154SEQ ID NO: 152 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 154 SEQ ID NO: 152 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 85 SEQ ID NO: 81 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 85 SEQ ID NO: 81(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 85SEQ ID NO: 81 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 162 SEQ ID NO: 177 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 162 SEQ ID NO: 177 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 162 SEQ ID NO: 177(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 89SEQ ID NO: 80 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 89 SEQ ID NO: 80 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 89 SEQ ID NO: 80 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 170 SEQ ID NO: 172(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 170SEQ ID NO: 172 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 170 SEQ ID NO: 172 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 75 SEQ ID NO: 196 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 75 SEQ ID NO: 196(CDR-H2) (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 75SEQ ID NO: 196 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 181 SEQ ID NO: 197 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 181 SEQ ID NO: 197 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 181 SEQ ID NO: 197(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 92SEQ ID NO: 198 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 92 SEQ ID NO: 198 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 92 SEQ ID NO: 198 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 184 SEQ ID NO: 199(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 184SEQ ID NO: 199 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 184 SEQ ID NO: 199 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 200 SEQ ID NO: 202 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 200 SEQ ID NO: 202(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 200SEQ ID NO: 202 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 201 SEQ ID NO: 203 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 201 SEQ ID NO: 203 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 201 SEQ ID NO: 203(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 204SEQ ID NO: 206 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 204 SEQ ID NO: 206 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 204 SEQ ID NO: 206 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 205 SEQ ID NO: 207(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 205SEQ ID NO: 207 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 205 SEQ ID NO: 207 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 208 SEQ ID NO: 210 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 208 SEQ ID NO: 210(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 208SEQ ID NO: 210 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 209 SEQ ID NO: 211 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 209 SEQ ID NO: 211 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 209 SEQ ID NO: 211(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 103SEQ ID NO: 126 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 103 SEQ ID NO: 126 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 103 SEQ ID NO: 126 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 104 SEQ ID NO: 127 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 104 SEQ ID NO: 127(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 104SEQ ID NO: 127 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 105SEQ ID NO: 128 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 105 SEQ ID NO: 128 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 105 SEQ ID NO: 128 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 106 SEQ ID NO: 129 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 106 SEQ ID NO: 129(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 106SEQ ID NO: 129 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 107SEQ ID NO: 130 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 107 SEQ ID NO: 130 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 107 SEQ ID NO: 130 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 108 SEQ ID NO: 131 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 108 SEQ ID NO: 131(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 108SEQ ID NO: 131 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 109SEQ ID NO: 132 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 109 SEQ ID NO: 132 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 109 SEQ ID NO: 132 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 110 SEQ ID NO: 133 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 110 SEQ ID NO: 133(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 110SEQ ID NO: 133 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 111SEQ ID NO: 134 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 111 SEQ ID NO: 134 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 111 SEQ ID NO: 134 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 112 SEQ ID NO: 135 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 112 SEQ ID NO: 135(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 112SEQ ID NO: 135 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 113SEQ ID NO: 136 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 113 SEQ ID NO: 136 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 113 SEQ ID NO: 136 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 114 SEQ ID NO: 137 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 114 SEQ ID NO: 137(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 114SEQ ID NO: 137 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 115SEQ ID NO: 138 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 115 SEQ ID NO: 138 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 115 SEQ ID NO: 138 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 116 SEQ ID NO: 139 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 116 SEQ ID NO: 139(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 116SEQ ID NO: 139 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 117SEQ ID NO: 140 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 117 SEQ ID NO: 140 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 117 SEQ ID NO: 140 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 118 SEQ ID NO: 141 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 118 SEQ ID NO: 141(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 118SEQ ID NO: 141 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 119SEQ ID NO: 142 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 119 SEQ ID NO: 142 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 119 SEQ ID NO: 142 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 120 SEQ ID NO: 143 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 120 SEQ ID NO: 143(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 120SEQ ID NO: 143 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 121SEQ ID NO: 144 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 121 SEQ ID NO: 144 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 121 SEQ ID NO: 144 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 122 SEQ ID NO: 145 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 122 SEQ ID NO: 145(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 122SEQ ID NO: 145 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 123SEQ ID NO: 146 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 123 SEQ ID NO: 146 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 123 SEQ ID NO: 146 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3); residues 31-35 ofresidues 31-35 of SEQ ID NO: 124 SEQ ID NO: 147 (CDR-H1); (CDR-H1);residues 50-66 of residues 50-66 of SEQ ID NO: 124 SEQ ID NO: 147(CDR-H2); (CDR-H2); residues 99-108 of residues 99-108 of SEQ ID NO: 124SEQ ID NO: 147 (CDR-H3); (CDR-H3); residues 24-34 of residues 24-34 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L1); (CDR-L1); residues 50-56 ofresidues 50-56 of SEQ ID NO: 59 SEQ ID NO: 59 (CDR-L2); (CDR-L2);residues 89-97 of residues 89-97 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L3); (CDR-L3); residues 31-35 of residues 31-35 of SEQ ID NO: 125SEQ ID NO: 148 (CDR-H1); (CDR-H1); residues 50-66 of residues 50-66 ofSEQ ID NO: 125 SEQ ID NO: 148 (CDR-H2); (CDR-H2); residues 99-108 ofresidues 99-108 of SEQ ID NO: 125 SEQ ID NO: 148 (CDR-H3); (CDR-H3);residues 24-34 of residues 24-34 of SEQ ID NO: 59 SEQ ID NO: 59(CDR-L1); (CDR-L1); residues 50-56 of residues 50-56 of SEQ ID NO: 59SEQ ID NO: 59 (CDR-L2); (CDR-L2); residues 89-97 of residues 89-97 ofSEQ ID NO: 59 SEQ ID NO: 59 (CDR-L3); (CDR-L3).

In another embodiment, a binding protein described above comprises atleast three CDRs, wherein the three CDRs are from a CDR set selectedfrom the group of CDR sets consisting of:

CDR Set SEQ ID NO. 1 residues 31-35 of SEQ ID NO: 60 (CDR-H1); residues50-66 of SEQ ID NO: 60 (CDR-H2); residues 99-108 of SEQ ID NO: 60(CDR-H3); 2 residues 24-34 of SEQ ID NO: 149 (CDR-L1); residues 50-56 ofSEQ ID NO: 149 (CDR-L2); residues 89-97 of SEQ ID NO: 149 (CDR-L3); 3residues 31-35 of SEQ ID NO: 69 (CDR-H1); residues 50-66 of SEQ ID NO:69 (CDR-H2); residues 99-108 of SEQ ID NO: 69 (CDR-H3); 4 residues 24-34of SEQ ID NO: 173 (CDR-L1); residues 50-56 of SEQ ID NO: 173 (CDR-L2);residues 89-97 of SEQ ID NO: 173 (CDR-L3); 5 residues 31-35 of SEQ IDNO: 67 (CDR-H1); residues 50-66 of SEQ ID NO: 67 (CDR-H2) residues99-108 of SEQ ID NO: 67 (CDR-H3); 6 residues 24-34 of SEQ ID NO: 151(CDR-L1); residues 50-56 of SEQ ID NO: 151 (CDR-L2); residues 89-97 ofSEQ ID NO: 151 (CDR-L3); 7 residues 31-35 of SEQ ID NO: 88 (CDR-H1);residues 50-66 of SEQ ID NO: 88 (CDR-H2); residues 99-108 of SEQ ID NO:88 (CDR-H3); 8 residues 24-34 of SEQ ID NO: 159 (CDR-L1); residues 50-56of SEQ ID NO: 159 (CDR-L2); residues 89-97 of SEQ ID NO: 159 (CDR-L3); 9residues 31-35 of SEQ ID NO: 96 (CDR-H1); residues 50-66 of SEQ ID NO:96 (CDR-H2); residues 99-108 of SEQ ID NO: 96 (CDR-H3); 10 residues24-34 of SEQ ID NO: 155 (CDR-L1); residues 50-56 of SEQ ID NO: 155(CDR-L2); residues 89-97 of SEQ ID NO: 155 (CDR-L3); 11 residues 31-35of SEQ ID NO: 90 (CDR-H1); residues 50-66 of SEQ ID NO: 90 (CDR-H2)residues 99-108 of SEQ ID NO: 90 (CDR-H3); 12 residues 24-34 of SEQ IDNO: 182 (CDR-L1); residues 50-56 of SEQ ID NO: 182 (CDR-L2); residues89-97 of SEQ ID NO: 182 (CDR-L3); 13 residues 31-35 of SEQ ID NO: 90(CDR-H1); residues 50-66 of SEQ ID NO: 90 (CDR-H2); residues 99-108 ofSEQ ID NO: 90 (CDR-H3); 14 residues 24-34 of SEQ ID NO: 167 (CDR-L1);residues 50-56 of SEQ ID NO: 167 (CDR-L2); residues 89-97 of SEQ ID NO:167 (CDR-L3); 15 residues 31-35 of SEQ ID NO: 90 (CDR-H1); residues50-66 of SEQ ID NO: 90 (CDR-H2); residues 99-108 of SEQ ID NO: 90(CDR-H3); 16 residues 24-34 of SEQ ID NO: 164 (CDR-L1); residues 50-56of SEQ ID NO: 164 (CDR-L2); residues 89-97 of SEQ ID NO: 164 (CDR-L3);17 residues 31-35 of SEQ ID NO: 73 (CDR-H1); residues 50-66 of SEQ IDNO: 73 (CDR-H2); residues 99-108 of SEQ ID NO: 73 (CDR-H3); 18 residues24-34 of SEQ ID NO: 174 (CDR-L1); residues 50-56 of SEQ ID NO: 174(CDR-L2); residues 89-97 of SEQ ID NO: 174 (CDR-L3); 19 residues 31-35of SEQ ID NO: 86 (CDR-H1); residues 50-66 of SEQ ID NO: 86 (CDR-H2);residues 99-108 of SEQ ID NO: 86 (CDR-H3); 20 residues 24-34 of SEQ IDNO: 159 (CDR-L1); residues 50-56 of SEQ ID NO: 159 (CDR-L2); residues89-97 of SEQ ID NO: 159 (CDR-L3); 21 residues 31-35 of SEQ ID NO: 90(CDR-H1); residues 50-66 of SEQ ID NO: 90 (CDR-H2); residues 99-108 ofSEQ ID NO: 90 (CDR-H3); 22 residues 24-34 of SEQ ID NO: 169 (CDR-L1);residues 50-56 of SEQ ID NO: 169 (CDR-L2); residues 89-97 of SEQ ID NO:169 (CDR-L3); 23 residues 31-35 of SEQ ID NO: 91 (CDR-H1); residues50-66 of SEQ ID NO: 91 (CDR-H2) residues 99-108 of SEQ ID NO: 91(CDR-H3); 24 residues 24-34 of SEQ ID NO: 165 (CDR-L1); residues 50-56of SEQ ID NO: 165 (CDR-L2); residues 89-97 of SEQ ID NO: 165 (CDR-L3);25 residues 31-35 of SEQ ID NO: 76 (CDR-H1); residues 50-66 of SEQ IDNO: 76 (CDR-H2); residues 99-108 of SEQ ID NO: 76 (CDR-H3); 26 residues24-34 of SEQ ID NO: 175 (CDR-L1); residues 50-56 of SEQ ID NO: 175(CDR-L2); residues 89-97 of SEQ ID NO: 175 (CDR-L3); 27 residues 31-35of SEQ ID NO: 99 (CDR-H1); residues 50-66 of SEQ ID NO: 99 (CDR-H2);residues 99-108 of SEQ ID NO: 99 (CDR-H3); 28 residues 24-34 of SEQ IDNO: 189 (CDR-L1); residues 50-56 of SEQ ID NO: 189 (CDR-L2); residues89-97 of SEQ ID NO: 189 (CDR-L3); 29 residues 31-35 of SEQ ID NO: 100(CDR-H1); residues 50-66 of SEQ ID NO: 100 (CDR-H2); residues 99-108 ofSEQ ID NO: 100 (CDR-H3); 30 residues 24-34 of SEQ ID NO: 168 (CDR-L1);residues 50-56 of SEQ ID NO: 168 (CDR-L2); residues 89-97 of SEQ ID NO:168 (CDR-L3); 31 residues 31-35 of SEQ ID NO: 71 (CDR-H1); residues50-66 of SEQ ID NO: 71 (CDR-H2) residues 99-108 of SEQ ID NO: 71(CDR-H3); 32 residues 24-34 of SEQ ID NO: 170 (CDR-L1); residues 50-56of SEQ ID NO: 170 (CDR-L2); residues 89-97 of SEQ ID NO: 170 (CDR-L3);33 residues 31-35 of SEQ ID NO: 84 (CDR-H1); residues 50-66 of SEQ IDNO: 84 (CDR-H2); residues 99-108 of SEQ ID NO: 84 (CDR-H3); 34 residues24-34 of SEQ ID NO: 188 (CDR-L1); residues 50-56 of SEQ ID NO: 188(CDR-L2); residues 89-97 of SEQ ID NO: 188 (CDR-L3); 35 residues 31-35of SEQ ID NO: 78 (CDR-H1); residues 50-66 of SEQ ID NO: 78 (CDR-H2);residues 99-108 of SEQ ID NO: 78 (CDR-H3); 36 residues 24-34 of SEQ IDNO: 179 (CDR-L1); residues 50-56 of SEQ ID NO: 179 (CDR-L2); residues89-97 of SEQ ID NO: 179 (CDR-L3); 37 residues 31-35 of SEQ ID NO: 93(CDR-H1); residues 50-66 of SEQ ID NO: 93 (CDR-H2); residues 99-108 ofSEQ ID NO: 93 (CDR-H3); 38 residues 24-34 of SEQ ID NO: 176 (CDR-L1);residues 50-56 of SEQ ID NO: 176 (CDR-L2); residues 89-97 of SEQ ID NO:176 (CDR-L3); 39 residues 31-35 of SEQ ID NO: 87 (CDR-H1); residues50-66 of SEQ ID NO: 87 (CDR-H2) residues 99-108 of SEQ ID NO: 87(CDR-H3); 40 residues 24-34 of SEQ ID NO: 154 (CDR-L1); residues 50-56of SEQ ID NO: 154 (CDR-L2); residues 89-97 of SEQ ID NO: 154 (CDR-L3);41 residues 31-35 of SEQ ID NO: 85 (CDR-H1); residues 50-66 of SEQ IDNO: 85 (CDR-H2); residues 99-108 of SEQ ID NO: 85 (CDR-H3); 42 residues24-34 of SEQ ID NO: 162 (CDR-L1); residues 50-56 of SEQ ID NO: 162(CDR-L2); residues 89-97 of SEQ ID NO: 162 (CDR-L3); 43 residues 31-35of SEQ ID NO: 89 (CDR-H1); residues 50-66 of SEQ ID NO: 89 (CDR-H2);residues 99-108 of SEQ ID NO: 89 (CDR-H3); 44 residues 24-34 of SEQ IDNO: 170 (CDR-L1); residues 50-56 of SEQ ID NO: 170 (CDR-L2); residues89-97 of SEQ ID NO: 170 (CDR-L3); 45 residues 31-35 of SEQ ID NO: 75(CDR-H1); residues 50-66 of SEQ ID NO: 75 (CDR-H2) residues 99-108 ofSEQ ID NO: 75 (CDR-H3); 46 residues 24-34 of SEQ ID NO: 181 (CDR-L1);residues 50-56 of SEQ ID NO: 181 (CDR-L2); residues 89-97 of SEQ ID NO:181 (CDR-L3); 47 residues 31-35 of SEQ ID NO: 92 (CDR-H1); residues50-66 of SEQ ID NO: 92 (CDR-H2); residues 99-108 of SEQ ID NO: 92(CDR-H3); 48 residues 24-34 of SEQ ID NO: 184 (CDR-L1); residues 50-56of SEQ ID NO: 184 (CDR-L2); residues 89-97 of SEQ ID NO: 184 (CDR-L3);49 residues 31-35 of SEQ ID NO: 200 (CDR-H1); residues 50-66 of SEQ IDNO: 200 (CDR-H2); residues 99-108 of SEQ ID NO: 200 (CDR-H3); 50residues 24-34 of SEQ ID NO: 201 (CDR-L1); residues 50-56 of SEQ ID NO:201 (CDR-L2); residues 89-97 of SEQ ID NO: 201 (CDR-L3); 51 residues31-35 of SEQ ID NO: 204 (CDR-H1); residues 50-66 of SEQ ID NO: 204(CDR-H2); residues 99-108 of SEQ ID NO: 204 (CDR-H3); 52 residues 24-34of SEQ ID NO: 205 (CDR-L1); residues 50-56 of SEQ ID NO: 205 (CDR-L2);residues 89-97 of SEQ ID NO: 205 (CDR-L3); 53 residues 31-35 of SEQ IDNO: 208 (CDR-H1); residues 50-66 of SEQ ID NO: 208 (CDR-H2); residues99-108 of SEQ ID NO: 208 (CDR-H3); 54 residues 24-34 of SEQ ID NO: 209(CDR-L1); residues 50-56 of SEQ ID NO: 209 (CDR-L2); residues 89-97 ofSEQ ID NO: 209 (CDR-L3); 55 residues 31-35 of SEQ ID NO: 63 (CDR-H1);residues 50-66 of SEQ ID NO: 63 (CDR-H2); residues 99-108 of SEQ ID NO:63 (CDR-H3); 56 residues 24-34 of SEQ ID NO: 150 (CDR-L1); residues50-56 of SEQ ID NO: 150 (CDR-L2); residues 89-97 of SEQ ID NO: 150(CDR-L3); 57 residues 31-35 of SEQ ID NO: 97 (CDR-H1); residues 50-66 ofSEQ ID NO: 97 (CDR-H2); residues 99-108 of SEQ ID NO: 97 (CDR-H3); 58residues 24-34 of SEQ ID NO: 187 (CDR-L1); residues 50-56 of SEQ ID NO:187 (CDR-L2); residues 89-97 of SEQ ID NO: 187 (CDR-L3); 59 residues31-35 of SEQ ID NO: 90 (CDR-H1); residues 50-66 of SEQ ID NO: 90(CDR-H2) residues 99-108 of SEQ ID NO: 90 (CDR-H3); 60 residues 24-34 ofSEQ ID NO: 156 (CDR-L1); residues 50-56 of SEQ ID NO: 156 (CDR-L2);residues 89-97 of SEQ ID NO: 156 (CDR-L3); 61 residues 31-35 of SEQ IDNO: 92 (CDR-H1); residues 50-66 of SEQ ID NO: 92 (CDR-H2); residues99-108 of SEQ ID NO: 92 (CDR-H3); 62 residues 24-34 of SEQ ID NO: 153(CDR-L1); residues 50-56 of SEQ ID NO: 153 (CDR-L2); residues 89-97 ofSEQ ID NO: 153 (CDR-L3); 63 residues 31-35 of SEQ ID NO: 94 (CDR-H1);residues 50-66 of SEQ ID NO: 94 (CDR-H2); residues 99-108 of SEQ ID NO:94 (CDR-H3); 64 residues 24-34 of SEQ ID NO: 166 (CDR-L1); residues50-56 of SEQ ID NO: 166 (CDR-L2); residues 89-97 of SEQ ID NO: 166(CDR-L3); 65 residues 31-35 of SEQ ID NO: 82 (CDR-H1); residues 50-66 ofSEQ ID NO: 82 (CDR-H2) residues 99-108 of SEQ ID NO: 82 (CDR-H3); 66residues 24-34 of SEQ ID NO: 159 (CDR-L1); residues 50-56 of SEQ ID NO:159 (CDR-L2); residues 89-97 of SEQ ID NO: 159 (CDR-L3); 67 residues31-35 of SEQ ID NO: 101 (CDR-H1); residues 50-66 of SEQ ID NO: 101(CDR-H2); residues 99-108 of SEQ ID NO: 101 (CDR-H3); 68 residues 24-34of SEQ ID NO: 158 (CDR-L1); residues 50-56 of SEQ ID NO: 158 (CDR-L2);residues 89-97 of SEQ ID NO: 158 (CDR-L3); 69 residues 31-35 of SEQ IDNO: 96 (CDR-H1); residues 50-66 of SEQ ID NO: 96 (CDR-H2); residues99-108 of SEQ ID NO: 96 (CDR-H3); 70 residues 24-34 of SEQ ID NO: 157(CDR-L1); residues 50-56 of SEQ ID NO: 157 (CDR-L2); residues 89-97 ofSEQ ID NO: 157 (CDR-L3); 71 residues 31-35 of SEQ ID NO: 70 (CDR-H1);residues 50-66 of SEQ ID NO: 70 (CDR-H2) residues 99-108 of SEQ ID NO:70 (CDR-H3); 72 residues 24-34 of SEQ ID NO: 161 (CDR-L1); residues50-56 of SEQ ID NO: 161 (CDR-L2); residues 89-97 of SEQ ID NO: 161(CDR-L3); 73 residues 31-35 of SEQ ID NO: 83 (CDR-H1); residues 50-66 ofSEQ ID NO: 83 (CDR-H2); residues 99-108 of SEQ ID NO: 83 (CDR-H3); 74residues 24-34 of SEQ ID NO: 158 (CDR-L1); residues 50-56 of SEQ ID NO:158 (CDR-L2); residues 89-97 of SEQ ID NO: 158 (CDR-L3); 75 residues31-35 of SEQ ID NO: 102 (CDR-H1); residues 50-66 of SEQ ID NO: 102(CDR-H2); residues 99-108 of SEQ ID NO: 102 (CDR-H3); 76 residues 24-34of SEQ ID NO: 185 (CDR-L1); residues 50-56 of SEQ ID NO: 185 (CDR-L2);residues 89-97 of SEQ ID NO: 185 (CDR-L3); 77 residues 31-35 of SEQ IDNO: 98 (CDR-H1); residues 50-66 of SEQ ID NO: 98 (CDR-H2); residues99-108 of SEQ ID NO: 98 (CDR-H3); 78 residues 24-34 of SEQ ID NO: 160(CDR-L1); residues 50-56 of SEQ ID NO: 160 (CDR-L2); residues 89-97 ofSEQ ID NO: 160 (CDR-L3); 79 residues 31-35 of SEQ ID NO: 74 (CDR-H1);residues 50-66 of SEQ ID NO: 74 (CDR-H2); residues 99-108 of SEQ ID NO:74 (CDR-H3); 80 residues 24-34 of SEQ ID NO: 186 (CDR-L1); residues50-56 of SEQ ID NO: 186 (CDR-L2); residues 89-97 of SEQ ID NO: 186(CDR-L3); 81 residues 31-35 of SEQ ID NO: 95 (CDR-H1); residues 50-66 ofSEQ ID NO: 95 (CDR-H2); residues 99-108 of SEQ ID NO: 95 (CDR-H3); 82residues 24-34 of SEQ ID NO: 157 (CDR-L1); residues 50-56 of SEQ ID NO:157 (CDR-L2); residues 89-97 of SEQ ID NO: 157 (CDR-L3); 83 residues31-35 of SEQ ID NO: 72 (CDR-H1); residues 50-66 of SEQ ID NO: 72(CDR-H2); residues 99-108 of SEQ ID NO: 72 (CDR-H3); 84 residues 24-34of SEQ ID NO: 178 (CDR-L1); residues 50-56 of SEQ ID NO: 178 (CDR-L2);residues 89-97 of SEQ ID NO: 178 (CDR-L3); 85 residues 31-35 of SEQ IDNO: 96 (CDR-H1); residues 50-66 of SEQ ID NO: 96 (CDR-H2); residues99-108 of SEQ ID NO: 96 (CDR-H3); 86 residues 24-34 of SEQ ID NO: 163(CDR-L1); residues 50-56 of SEQ ID NO: 163 (CDR-L2); residues 89-97 ofSEQ ID NO: 163 (CDR-L3); 87 residues 31-35 of SEQ ID NO: 77 (CDR-H1);residues 50-66 of SEQ ID NO: 77 (CDR-H2); residues 99-108 of SEQ ID NO:77 (CDR-H3); 88 residues 24-34 of SEQ ID NO: 183 (CDR-L1); residues50-56 of SEQ ID NO: 183 (CDR-L2); residues 89-97 of SEQ ID NO: 183(CDR-L3); 89 residues 31-35 of SEQ ID NO: 90 (CDR-H1); residues 50-66 ofSEQ ID NO: 90 (CDR-H2); residues 99-108 of SEQ ID NO: 90 (CDR-H3); 90residues 24-34 of SEQ ID NO: 171 (CDR-L1); residues 50-56 of SEQ ID NO:171 (CDR-L2); residues 89-97 of SEQ ID NO: 171 (CDR-L3); 91 residues31-35 of SEQ ID NO: 79 (CDR-H1); residues 50-66 of SEQ ID NO: 79(CDR-H2); residues 99-108 of SEQ ID NO: 79 (CDR-H3); 92 residues 24-34of SEQ ID NO: 180 (CDR-L1); residues 50-56 of SEQ ID NO: 180 (CDR-L2);residues 89-97 of SEQ ID NO: 180 (CDR-L3); 93 residues 31-35 of SEQ IDNO: 381 (CDR-H1); residues 50-66 of SEQ ID NO: 381 (CDR-H2); residues99-108 of SEQ ID NO: 381 (CDR-H3); 94 residues 24-34 of SEQ ID NO: 152(CDR-L1); residues 50-56 of SEQ ID NO: 152 (CDR-L2); residues 89-97 ofSEQ ID NO: 152 (CDR-L3); 95 residues 31-35 of SEQ ID NO: 81 (CDR-H1);residues 50-66 of SEQ ID NO: 81 (CDR-H2); residues 99-108 of SEQ ID NO:81 (CDR-H3); 96 residues 24-34 of SEQ ID NO: 177 (CDR-L1); residues50-56 of SEQ ID NO: 177 (CDR-L2); residues 89-97 of SEQ ID NO: 177(CDR-L3); 97 residues 31-35 of SEQ ID NO: 80 (CDR-H1); residues 50-66 ofSEQ ID NO: 80 (CDR-H2); residues 99-108 of SEQ ID NO: 80 (CDR-H3); 98residues 24-34 of SEQ ID NO: 172 (CDR-L1); residues 50-56 of SEQ ID NO:172 (CDR-L2); residues 89-97 of SEQ ID NO: 172 (CDR-L3); 99 residues31-35 of SEQ ID NO: 196 (CDR-H1); residues 50-66 of SEQ ID NO: 196(CDR-H2); residues 99-108 of SEQ ID NO: 196 (CDR-H3); 100 residues 24-34of SEQ ID NO: 197 (CDR-L1); residues 50-56 of SEQ ID NO: 197 (CDR-L2);residues 89-97 of SEQ ID NO: 197 (CDR-L3); 101 residues 31-35 of SEQ IDNO: 198 (CDR-H1); residues 50-66 of SEQ ID NO: 198 (CDR-H2); residues99-108 of SEQ ID NO: 198 (CDR-H3); 102 residues 24-34 of SEQ ID NO: 199(CDR-L1); residues 50-56 of SEQ ID NO: 199 (CDR-L2); residues 89-97 ofSEQ ID NO: 199 (CDR-L3); 103 residues 31-35 of SEQ ID NO: 202 (CDR-H1);residues 50-66 of SEQ ID NO: 202 (CDR-H2); residues 99-108 of SEQ ID NO:202 (CDR-H3); 104 residues 24-34 of SEQ ID NO: 203 (CDR-L1); residues50-56 of SEQ ID NO: 203 (CDR-L2); residues 89-97 of SEQ ID NO: 203(CDR-L3); 105 residues 31-35 of SEQ ID NO: 206 (CDR-H1); residues 50-66of SEQ ID NO: 206 (CDR-H2); residues 99-108 of SEQ ID NO: 206 (CDR-H3);106 residues 24-34 of SEQ ID NO: 207 (CDR-L1); residues 50-56 of SEQ IDNO: 207 (CDR-L2); residues 89-97 of SEQ ID NO: 207 (CDR-L3); 107residues 31-35 of SEQ ID NO: 210 (CDR-H1); residues 50-66 of SEQ ID NO:210 (CDR-H2); residues 99-108 of SEQ ID NO: 210 (CDR-H3); 108 residues24-34 of SEQ ID NO: 211 (CDR-L1); residues 50-56 of SEQ ID NO: 211(CDR-L2); residues 89-97 of SEQ ID NO: 211 (CDR-L3); 109 residues 31-35of SEQ ID NO: 103 (CDR-H1); residues 50-66 of SEQ ID NO: 103 (CDR-H2);residues 99-108 of SEQ ID NO: 103 (CDR-H3); 110 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 111 residues 31-35 of SEQ ID NO: 104(CDR-H1); residues 50-66 of SEQ ID NO: 104 (CDR-H2); residues 99-108 ofSEQ ID NO: 104 (CDR-H3); 112 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 113 residues 31-35 of SEQ ID NO: 105 (CDR-H1); residues50-66 of SEQ ID NO: 105 (CDR-H2); residues 99-108 of SEQ ID NO: 105(CDR-H3); 114 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 115residues 31-35 of SEQ ID NO: 106 (CDR-H1); residues 50-66 of SEQ ID NO:106 (CDR-H2); residues 99-108 of SEQ ID NO: 106 (CDR-H3); 116 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 117 residues 31-35of SEQ ID NO: 107 (CDR-H1); residues 50-66 of SEQ ID NO: 107 (CDR-H2);residues 99-108 of SEQ ID NO: 107 (CDR-H3); 118 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 119 residues 31-35 of SEQ ID NO: 108(CDR-H1); residues 50-66 of SEQ ID NO: 108 (CDR-H2); residues 99-108 ofSEQ ID NO: 108 (CDR-H3); 120 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 121 residues 31-35 of SEQ ID NO: 109 (CDR-H1); residues50-66 of SEQ ID NO: 109 (CDR-H2); residues 99-108 of SEQ ID NO: 109(CDR-H3); 122 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 123residues 31-35 of SEQ ID NO: 110 (CDR-H1); residues 50-66 of SEQ ID NO:110 (CDR-H2); residues 99-108 of SEQ ID NO: 110 (CDR-H3); 124 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 125 residues 31-35of SEQ ID NO: 111 (CDR-H1); residues 50-66 of SEQ ID NO: 111 (CDR-H2);residues 99-108 of SEQ ID NO: 111 (CDR-H3); 126 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 127 residues 31-35 of SEQ ID NO: 112(CDR-H1); residues 50-66 of SEQ ID NO: 112 (CDR-H2); residues 99-108 ofSEQ ID NO: 112 (CDR-H3); 128 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 129 residues 31-35 of SEQ ID NO: 113 (CDR-H1); residues50-66 of SEQ ID NO: 113 (CDR-H2); residues 99-108 of SEQ ID NO: 113(CDR-H3); 130 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 131residues 31-35 of SEQ ID NO: 114 (CDR-H1); residues 50-66 of SEQ ID NO:114 (CDR-H2); residues 99-108 of SEQ ID NO: 114 (CDR-H3); 132 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 133 residues 31-35of SEQ ID NO: 115 (CDR-H1); residues 50-66 of SEQ ID NO: 115 (CDR-H2);residues 99-108 of SEQ ID NO: 115 (CDR-H3); 134 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 135 residues 31-35 of SEQ ID NO: 116(CDR-H1); residues 50-66 of SEQ ID NO: 116 (CDR-H2); residues 99-108 ofSEQ ID NO: 116 (CDR-H3); 136 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 137 residues 31-35 of SEQ ID NO: 117 (CDR-H1); residues50-66 of SEQ ID NO: 117 (CDR-H2); residues 99-108 of SEQ ID NO: 117(CDR-H3); 138 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 139residues 31-35 of SEQ ID NO: 118 (CDR-H1); residues 50-66 of SEQ ID NO:118 (CDR-H2); residues 99-108 of SEQ ID NO: 118 (CDR-H3); 140 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 141 residues 31-35of SEQ ID NO: 119 (CDR-H1); residues 50-66 of SEQ ID NO: 119 (CDR-H2);residues 99-108 of SEQ ID NO: 119 (CDR-H3); 142 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 143 residues 31-35 of SEQ ID NO: 120(CDR-H1); residues 50-66 of SEQ ID NO: 120 (CDR-H2); residues 99-108 ofSEQ ID NO: 120 (CDR-H3); 144 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 145 residues 31-35 of SEQ ID NO: 121 (CDR-H1); residues50-66 of SEQ ID NO: 121 (CDR-H2); residues 99-108 of SEQ ID NO: 121(CDR-H3); 146 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 147residues 31-35 of SEQ ID NO: 122 (CDR-H1); residues 50-66 of SEQ ID NO:122 (CDR-H2); residues 99-108 of SEQ ID NO: 122 (CDR-H3); 148 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 149 residues 31-35of SEQ ID NO: 123 (CDR-H1); residues 50-66 of SEQ ID NO: 123 (CDR-H2);residues 99-108 of SEQ ID NO: 123 (CDR-H3); 150 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 151 residues 31-35 of SEQ ID NO: 124(CDR-H1); residues 50-66 of SEQ ID NO: 124 (CDR-H2); residues 99-108 ofSEQ ID NO: 124 (CDR-H3); 152 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 153 residues 31-35 of SEQ ID NO: 125 (CDR-H1); residues50-66 of SEQ ID NO: 125 (CDR-H2); residues 99-108 of SEQ ID NO: 125(CDR-H3); 154 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 155residues 31-35 of SEQ ID NO: 126 (CDR-H1); residues 50-66 of SEQ ID NO:126 (CDR-H2); residues 99-108 of SEQ ID NO: 126 (CDR-H3); 156 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 157 residues 31-35of SEQ ID NO: 127 (CDR-H1); residues 50-66 of SEQ ID NO: 127 (CDR-H2);residues 99-108 of SEQ ID NO: 127 (CDR-H3); 158 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 159 residues 31-35 of SEQ ID NO: 128(CDR-H1); residues 50-66 of SEQ ID NO: 128 (CDR-H2); residues 99-108 ofSEQ ID NO: 128 (CDR-H3); 160 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 161 residues 31-35 of SEQ ID NO: 129 (CDR-H1); residues50-66 of SEQ ID NO: 129 (CDR-H2); residues 99-108 of SEQ ID NO: 129(CDR-H3); 162 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 163residues 31-35 of SEQ ID NO: 130 (CDR-H1); residues 50-66 of SEQ ID NO:130 (CDR-H2); residues 99-108 of SEQ ID NO: 130 (CDR-H3); 164 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 165 residues 31-35of SEQ ID NO: 131 (CDR-H1); residues 50-66 of SEQ ID NO: 131 (CDR-H2);residues 99-108 of SEQ ID NO: 131 (CDR-H3); 166 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 167 residues 31-35 of SEQ ID NO: 132(CDR-H1); residues 50-66 of SEQ ID NO: 132 (CDR-H2); residues 99-108 ofSEQ ID NO: 132 (CDR-H3); 168 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 169 residues 31-35 of SEQ ID NO: 133 (CDR-H1); residues50-66 of SEQ ID NO: 133 (CDR-H2); residues 99-108 of SEQ ID NO: 133(CDR-H3); 170 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 171residues 31-35 of SEQ ID NO: 134 (CDR-H1); residues 50-66 of SEQ ID NO:134 (CDR-H2); residues 99-108 of SEQ ID NO: 134 (CDR-H3); 172 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 173 residues 31-35of SEQ ID NO: 135 (CDR-H1); residues 50-66 of SEQ ID NO: 135 (CDR-H2);residues 99-108 of SEQ ID NO: 135 (CDR-H3); 174 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 175 residues 31-35 of SEQ ID NO: 136(CDR-H1); residues 50-66 of SEQ ID NO: 136 (CDR-H2); residues 99-108 ofSEQ ID NO: 136 (CDR-H3); 176 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 177 residues 31-35 of SEQ ID NO: 137 (CDR-H1); residues50-66 of SEQ ID NO: 137 (CDR-H2); residues 99-108 of SEQ ID NO: 137(CDR-H3); 178 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 179residues 31-35 of SEQ ID NO: 138 (CDR-H1); residues 50-66 of SEQ ID NO:138 (CDR-H2); residues 99-108 of SEQ ID NO: 138 (CDR-H3); 180 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 181 residues 31-35of SEQ ID NO: 139 (CDR-H1); residues 50-66 of SEQ ID NO: 139 (CDR-H2);residues 99-108 of SEQ ID NO: 139 (CDR-H3); 182 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 183 residues 31-35 of SEQ ID NO: 140(CDR-H1); residues 50-66 of SEQ ID NO: 140 (CDR-H2); residues 99-108 ofSEQ ID NO: 140 (CDR-H3); 184 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 185 residues 31-35 of SEQ ID NO: 141 (CDR-H1); residues50-66 of SEQ ID NO: 141 (CDR-H2); residues 99-108 of SEQ ID NO: 141(CDR-H3); 186 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 187residues 31-35 of SEQ ID NO: 142 (CDR-H1); residues 50-66 of SEQ ID NO:142 (CDR-H2); residues 99-108 of SEQ ID NO: 142 (CDR-H3); 188 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 189 residues 31-35of SEQ ID NO: 143 (CDR-H1); residues 50-66 of SEQ ID NO: 143 (CDR-H2);residues 99-108 of SEQ ID NO: 143 (CDR-H3); 190 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 191 residues 31-35 of SEQ ID NO: 144(CDR-H1); residues 50-66 of SEQ ID NO: 144 (CDR-H2); residues 99-108 ofSEQ ID NO: 144 (CDR-H3); 192 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3); 193 residues 31-35 of SEQ ID NO: 145 (CDR-H1); residues50-66 of SEQ ID NO: 145 (CDR-H2); residues 99-108 of SEQ ID NO: 145(CDR-H3); 194 residues 24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 195residues 31-35 of SEQ ID NO: 146 (CDR-H1); residues 50-66 of SEQ ID NO:146 (CDR-H2); residues 99-108 of SEQ ID NO: 146 (CDR-H3); 196 residues24-34 of SEQ ID NO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59(CDR-L2); residues 89-97 of SEQ ID NO: 59 (CDR-L3); 197 residues 31-35of SEQ ID NO: 147 (CDR-H1); residues 50-66 of SEQ ID NO: 147 (CDR-H2);residues 99-108 of SEQ ID NO: 147 (CDR-H3); 198 residues 24-34 of SEQ IDNO: 59 (CDR-L1); residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues89-97 of SEQ ID NO: 59 (CDR-L3); 199 residues 31-35 of SEQ ID NO: 148(CDR-H1); residues 50-66 of SEQ ID NO: 148 (CDR-H2); residues 99-108 ofSEQ ID NO: 148 (CDR-H3); 200 residues 24-34 of SEQ ID NO: 59 (CDR-L1);residues 50-56 of SEQ ID NO: 59 (CDR-L2); residues 89-97 of SEQ ID NO:59 (CDR-L3).

In an embodiment, a binding protein described above comprises CDRs fromtwo CDR sets selected from the above group of CDR sets.

In another embodiment, the invention provides a binding proteincomprising CDRs from two CDR sets from the group above, wherein the twoCDR sets are selected from the group consisting of:

CDR Set 1 and CDR Set 2 CDR Set 3 and CDR Set 4 CDR Set 5 and CDR Set 6CDR Set 7 and CDR Set 8 CDR Set 9 and CDR Set 10 CDR Set 11 and CDR Set12 CDR Set 13 and CDR Set 14 CDR Set 15 and CDR Set 16 CDR Set 17 andCDR Set 18 CDR Set 19 and CDR Set 20 CDR Set 21 and CDR Set 22 CDR Set23 and CDR Set 24 CDR Set 25 and CDR Set 26 CDR Set 27 and CDR Set 28CDR Set 29 and CDR Set 30 CDR Set 31 and CDR Set 32 CDR Set 33 and CDRSet 34 CDR Set 35 and CDR Set 36 CDR Set 37 and CDR Set 38 CDR Set 39and CDR Set 40 CDR Set 41 and CDR Set 42 CDR Set 43 and CDR Set 44 CDRSet 45 and CDR Set 46 CDR Set 47 and CDR Set 48 CDR Set 49 and CDR Set50 CDR Set 51 and CDR Set 52 CDR Set 53 and CDR Set 54 CDR Set 55 andCDR Set 56 CDR Set 57 and CDR Set 58 CDR Set 59 and CDR Set 60 CDR Set61 and CDR Set 62 CDR Set 63 and CDR Set 64 CDR Set 65 and CDR Set 66CDR Set 67 and CDR Set 68 CDR Set 69 and CDR Set 70 CDR Set 71 and CDRSet 72 CDR Set 73 and CDR Set 74 CDR Set 75 and CDR Set 76 CDR Set 77and CDR Set 78 CDR Set 79 and CDR Set 80 CDR Set 81 and CDR Set 82 CDRSet 83 and CDR Set 84 CDR Set 85 and CDR Set 86 CDR Set 87 and CDR Set88 CDR Set 89 and CDR Set 90 CDR Set 91 and CDR Set 92 CDR Set 93 andCDR Set 94 CDR Set 95 and CDR Set 96 CDR Set 97 and CDR Set 98 CDR Set99 and CDR Set 100 CDR Set 101 and CDR Set 102 CDR Set 103 and CDR Set104 CDR Set 105 and CDR Set 106 CDR Set 107 and CDR Set 108 CDR Set 109and CDR Set 110 CDR Set 111 and CDR Set 112 CDR Set 113 and CDR Set 114CDR Set 115 and CDR Set 116 CDR Set 117 and CDR Set 118 CDR Set 119 andCDR Set 120 CDR Set 121 and CDR Set 122 CDR Set 123 and CDR Set 124 CDRSet 125 and CDR Set 126 CDR Set 127 and CDR Set 128 CDR Set 129 and CDRSet 130 CDR Set 131 and CDR Set 132 CDR Set 133 and CDR Set 134 CDR Set135 and CDR Set 136 CDR Set 137 and CDR Set 138 CDR Set 139 and CDR Set140 CDR Set 141 and CDR Set 142 CDR Set 143 and CDR Set 144 CDR Set 145and CDR Set 146 CDR Set 147 and CDR Set 148 CDR Set 149 and CDR Set 150CDR Set 151 and CDR Set 152 CDR Set 153 and CDR Set 154 CDR Set 155 andCDR Set 156 CDR Set 157 and CDR Set 158 CDR Set 159 and CDR Set 160 CDRSet 161 and CDR Set 162 CDR Set 163 and CDR Set 164 CDR Set 165 and CDRSet 166 CDR Set 167 and CDR Set 168 CDR Set 169 and CDR Set 170 CDR Set171 and CDR Set 172 CDR Set 173 and CDR Set 174 CDR Set 175 and CDR Set176 CDR Set 177 and CDR Set 178 CDR Set 179 and CDR Set 180 CDR Set 181and CDR Set 182 CDR Set 183 and CDR Set 184 CDR Set 185 and CDR Set 186CDR Set 187 and CDR Set 188 CDR Set 189 and CDR Set 190 CDR Set 191 andCDR Set 192 CDR Set 193 and CDR Set 194 CDR Set 195 and CDR Set 196 CDRSet 197 and CDR Set 198 CDR Set 199 and CDR Set 200

In another embodiment, a binding protein described above furthercomprises a human acceptor framework. Preferably, the human frameworkcomprises an amino acid sequence selected group consisting SEQ ID NOs:7-10, 13-16, 25, 240-316, and 317-381. In an embodiment, a bindingprotein of the invention comprises a human framework sequence selectedfrom the group consisting of SEQ ID NOS:7-10 and 13-16.

Binding proteins of the invention include those that comprise a humanacceptor framework comprising at least one Framework Region amino acidsubstitution, wherein the amino acid sequence of the framework is atleast 65% identical to the sequence of said human acceptor framework andcomprises at least 70 amino acid residues identical to said humanacceptor framework.

In another embodiment, a binding protein of the invention comprises ahuman acceptor framework, wherein said acceptor framework comprises atleast one framework region amino acid substitution at a key residue,said key residue selected from the group consisting of a residueadjacent to a CDR; a glycosylation site residue; a rare residue; aresidue capable of interacting with human IL-1β; a residue capable ofinteracting with a CDR; a canonical residue; a contact residue betweenheavy chain variable region and light chain variable region; a residuewithin a Vernier zone; and a residue in a region that overlaps between aChothia-defined variable heavy chain CDR1 and a Kabat-defined firstheavy chain framework. In an exemplary embodiment, a binding protein ofthe invention comprises a key residue, wherein said key residue isselected from the group consisting of: 2H, 4H, 24H, 26H, 27H, 29H, 34H,35H, 37H, 39H, 44H, 45H, 47H, 48H, 49H, 50H, 51H, 58H, 59H, 60H, 63H,67H, 69H, 71H, 73H, 76H, 78H, 91H, 93H, 94H, 2L, 4L, 25L, 29L, 27L, 33L,34L, 36L, 38L, 43L, 44L, 46L, 47L, 48L, 49L, 55L, 58L, 62L, 64L, 71L,87L, 89L, 90L, 91L, 94L, 95L (all Kabat numbering). An exemplary subsetof these residues for the humanization of a binding protein of theinvention consists of 27H, 48H, 67H, 69H, 93H, 36L, 43L, 46L, 47L, 49L,58L, 71L, and 87L.

In another embodiment, a binding protein of the invention comprises aconsensus human variable domain.

In an embodiment, an IL-1β binding protein of the invention comprises atleast one variable domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs:60-189.

In another embodiment, a binding protein of the invention comprises atleast one variable heavy chain (VH) region (or domain) comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:60-148, 196, 198, 200, 202, 204, 206, 208, and 210.

In another embodiment, a binding protein according to the inventioncomprises at least one variable light chain (VL) region (or domain)comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:149-189.

In still another embodiment, a binding protein according to theinvention comprises at least one VH region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 60-148, 196,198, 200, 202, 204, 206, 208, and 210, and at least one VL regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:149-189, 197, 199, 201, 203, 205, 207, 209, and 211.

In an embodiment, a binding protein according to the invention comprisestwo variable domains, wherein the two variable domains comprise aminoacid sequences selected from the group consisting of:

SEQ ID NO:60 and SEQ ID NO:149 SEQ ID NO:198 and SEQ ID NO:199 SEQ IDNO:69 and SEQ ID NO:173 SEQ ID NO:202 and SEQ ID NO:203 SEQ ID NO:67 andSEQ ID NO:151 SEQ ID NO:206 and SEQ ID NO:207 SEQ ID NO:88 and SEQ IDNO:159 SEQ ID NO:210 and SEQ ID NO:211 SEQ ID NO:96 and SEQ ID NO:155SEQ ID NO:103 and SEQ ID NO:59 SEQ ID NO:90 and SEQ ID NO:182 SEQ IDNO:104 and SEQ ID NO:59 SEQ ID NO:90 and SEQ ID NO:167 SEQ ID NO:105 andSEQ ID NO:59 SEQ ID NO:90 and SEQ ID NO:164 SEQ ID NO:106 and SEQ IDNO:59 SEQ ID NO:73 and SEQ ID NO:174 SEQ ID NO:107 and SEQ ID NO:59 SEQID NO:86 and SEQ ID NO:159 SEQ ID NO:108 and SEQ ID NO:59 SEQ ID NO:90and SEQ ID NO:169 SEQ ID NO:109 and SEQ ID NO:59 SEQ ID NO:91 and SEQ IDNO:165 SEQ ID NO:110 and SEQ ID NO:59 SEQ ID NO:76 and SEQ ID NO:175 SEQID NO:111 and SEQ ID NO:59 SEQ ID NO:99 and SEQ ID NO:189 SEQ ID NO:112and SEQ ID NO:59 SEQ ID NO:100 and SEQ ID NO:168 SEQ ID NO:113 and SEQID NO:59 SEQ ID NO:71 and SEQ ID NO:170 SEQ ID NO:114 and SEQ ID NO:59SEQ ID NO:84 and SEQ ID NO:188 SEQ ID NO:115 and SEQ ID NO:59 SEQ IDNO:78 and SEQ ID NO:179 SEQ ID NO:116 and SEQ ID NO:59 SEQ ID NO:93 andSEQ ID NO:176 SEQ ID NO:117 and SEQ ID NO:59 SEQ ID NO:87 and SEQ IDNO:154 SEQ ID NO:118 and SEQ ID NO:59 SEQ ID NO:85 and SEQ ID NO:162 SEQID NO:119 and SEQ ID NO:59 SEQ ID NO:89 and SEQ ID NO:170 SEQ ID NO:120and SEQ ID NO:59 SEQ ID NO:75 and SEQ ID NO:181 SEQ ID NO:121 and SEQ IDNO:59 SEQ ID NO:92 and SEQ ID NO:184 SEQ ID NO:122 and SEQ ID NO:59 SEQID NO:200 and SEQ ID NO:201 SEQ ID NO:123 and SEQ ID NO:59 SEQ ID NO:204and SEQ ID NO:205 SEQ ID NO:124 and SEQ ID NO:59 SEQ ID NO:208 and SEQID NO:209 SEQ ID NO:125 and SEQ ID NO:59 SEQ ID NO:63 and SEQ ID NO:150SEQ ID NO:126 and SEQ ID NO:59 SEQ ID NO:97 and SEQ ID NO:187 SEQ IDNO:127 and SEQ ID NO:59 SEQ ID NO:90 and SEQ ID NO:156 SEQ ID NO:128 andSEQ ID NO:59 SEQ ID NO:92 and SEQ ID NO:153 SEQ ID NO:129 and SEQ IDNO:59 SEQ ID NO:94 and SEQ ID NO:166 SEQ ID NO:130 and SEQ ID NO:59 SEQID NO:82 and SEQ ID NO:159 SEQ ID NO:131 and SEQ ID NO:59 SEQ ID NO:101and SEQ ID NO:158 SEQ ID NO:132 and SEQ ID NO:59 SEQ ID NO:96 and SEQ IDNO:157 SEQ ID NO:133 and SEQ ID NO:59 SEQ ID NO:70 and SEQ ID NO:161 SEQID NO:134 and SEQ ID NO:59 SEQ ID NO:83 and SEQ ID NO:158 SEQ ID NO:135and SEQ ID NO:59 SEQ ID NO:102 and SEQ ID NO:185 SEQ ID NO:136 and SEQID NO:59 SEQ ID NO:98 and SEQ ID NO:160 SEQ ID NO:137 and SEQ ID NO:59SEQ ID NO:74 and SEQ ID NO:186 SEQ ID NO:138 and SEQ ID NO:59 SEQ IDNO:95 and SEQ ID NO:157 SEQ ID NO:139 and SEQ ID NO:59 SEQ ID NO:72 andSEQ ID NO:178 SEQ ID NO:140 and SEQ ID NO:59 SEQ ID NO:96 and SEQ IDNO:163 SEQ ID NO:141 and SEQ ID NO:59 SEQ ID NO:77 and SEQ ID NO:183 SEQID NO:142 and SEQ ID NO:59 SEQ ID NO:90 and SEQ ID NO:171 SEQ ID NO:143and SEQ ID NO:59 SEQ ID NO:79 and SEQ ID NO:180 SEQ ID NO:144 and SEQ IDNO:59 SEQ ID NO:381 and SEQ ID NO:152 SEQ ID NO:145 and SEQ ID NO:59 SEQID NO:81 and SEQ ID NO:177 SEQ ID NO:146 and SEQ ID NO:59 SEQ ID NO:80and SEQ ID NO:172 SEQ ID NO:147 and SEQ ID NO:59 SEQ ID NO:196 and SEQID NO:197 SEQ ID NO:148 and SEQ ID NO:59

In another embodiment, an IL-1β binding protein described herein isselected from the group consisting of: an immunoglobulin molecule, anscFv, a monoclonal antibody, a humanized antibody, a chimeric antibody,a humanized antibody, a Fab fragment, an Fab′ fragment, an F(ab′)₂, anFv, and a disulfide linked Fv.

In an aspect of the invention, a binding protein described herein iscapable of modulating a biological function of IL-1. In another aspect,a binding protein described herein is capable of neutralizing IL-1.

In an embodiment, a binding protein described herein has an on rateconstant (K_(on)) to IL-1β selected from the group consisting of: atleast about 10² M⁻¹ s⁻¹; at least about 10³ M⁻¹ s⁻¹; at least about 10⁴M⁻¹ s⁻¹; at least about 10⁵ M⁻¹ s⁻¹; and at least about 10⁶ M⁻¹ s⁻¹; asmeasured by surface plasmon resonance.

In another embodiment, a binding protein described herein has an offrate constant (K_(off)) to IL-1β selected from the group consisting of:at most about 10⁻³ s⁻¹; at most about 10⁻⁴ s⁻¹; at most about 10⁻⁵ s⁻¹;and at most about 10⁻⁶ s⁻¹, as measured by surface plasmon resonance.

In another embodiment, a binding protein described herein has adissociation constant (K_(D)) to IL-1β selected from the groupconsisting of: at most about 10⁻⁷ M; at most about 10⁻⁸ M; at most about10⁻⁹ M; at most about 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about10⁻¹² M; and at most 10⁻¹³ M.

In one aspect, the invention provides a binding protein construct thatcomprises a binding protein described herein and further comprises alinker or an immunoglobulin constant domain. In an embodiment, thebinding protein construct comprises a binding protein, wherein thebinding protein is selected from the group consisting of: animmunoglobulin molecule, a disulfide linked Fv, a monoclonal antibody,an scFv, a chimeric antibody, a CDR-grafted antibody, a diabody, ahumanized antibody, a multispecific antibody, an Fab, a dual specificantibody, a Fab′, a bispecific antibody, and a F(ab′)₂, a DVD-Ig™, andan Fv.

In an embodiment, a binding protein construct comprises a heavy chainimmunoglobulin constant domain selected from the group consisting of ahuman IgM constant domain, a human IgG4 constant domain, a human IgG1constant domain, a human IgE constant domain, a human IgG2 constantdomain, and a human IgG3 constant domain, and a human IgA constantdomain.

In another embodiment, a binding protein construct comprises animmunoglobulin constant domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, andSEQ ID NO:6.

The invention also provide a binding protein conjugate that comprises abinding protein construct described herein and further comprises anagent selected from the group consisting of an immunoadhesion molecule,an imaging agent, a therapeutic agent, and a cytotoxic agent. Imagingagents that are useful as agent moieties in binding protein conjugatesdescribed herein include, but are not limited to, a radiolabel, anenzyme, a fluorescent label, a luminescent label, a bioluminescentlabel, a magnetic label, and biotin. In an embodiment, a radiolabel isselected from the group consisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm.

In another embodiment, a binding protein conjugate comprises an agentthat is a therapeutic or cytotoxic agent selected from the groupconsisting of: an anti-metabolite, an alkylating agent, an antibiotic, agrowth factor, a cytokine, an anti-angiogenic agent, an anti-mitoticagent, an anthracycline, toxin, and an apoptotic agent.

In an embodiment, a binding protein, binding protein construct, orbinding protein conjugate described herein possesses a humanglycosylation pattern.

Binding proteins, binding protein constructs, and binding proteinconjugates described herein may exist as soluble proteins or ascrystals. In an embodiment, such crystals are carrier-freepharmaceutical controlled released crystals. In another embodiment, acrystalline form of a binding protein, binding protein construct, orbinding protein conjugate described herein has a greater in vivohalf-life than its soluble counterpart. In another embodiment, a crystalof a binding protein, binding protein construct, or binding proteinconjugate described herein retains biological activity of its solublecounterpart.

Compositions of the invention include a composition for the release of acrystallized binding protein, binding protein construct, or bindingprotein conjugate described herein, comprising:

(a) a formulation, wherein said formulation comprises a crystallizedbinding protein, binding protein construct, or binding protein conjugatedescribed herein, and an ingredient; and

(b) at least one polymeric carrier.

Polymeric carriers useful in compositions of the invention include,without limitation, one or more of the group consisting of: poly(acrylic acid), poly (cyanoacrylates), poly (amino acids), poly(anhydrides), poly (depsipeptide), poly (esters), poly (lactic acid),poly (lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly(caprolactone), poly (dioxanone); poly (ethylene glycol), poly((hydroxypropyl)methacrylamide, poly [(organo) phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof.

In another aspect, an ingredient of a composition of the invention isselected from the group consisting of albumin, sucrose, trehalose,lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethyleneglycol and polyethylene glycol.

The invention also provides pharmaceutical compositions comprising abinding protein, a binding protein construct, or binding proteinconjugate described herein, and a pharmaceutically acceptable carrier.Pharmaceutical compositions of the invention may further comprise atleast one additional agent. In an embodiment, such an additional agentincludes, but is not limited to, therapeutic agent, imaging agent,cytotoxic agent, angiogenesis inhibitors; kinase inhibitors;co-stimulation molecule blockers; adhesion molecule blockers;anti-cytokine antibody or functional fragment thereof; methotrexate;cyclosporin; rapamycin; FK506; detectable label or reporter; a TNFantagonist; an antirheumatic; a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid,an erythropoietin, an immunization, an immunoglobulin, animmunosuppressive, a growth hormone, a hormone replacement drug, aradiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,an asthma medication, a beta agonist, an inhaled steroid, an epinephrineor analog, a cytokine, and a cytokine antagonist.

In an embodiment, a pharmaceutical composition of the inventioncomprises a pharmaceutically acceptable carrier, wherein the carrieralso serves as an adjuvant to increase the absorption or dispersion ofthe binding protein, binding protein construct, or binding proteinconjugate in the composition. An exemplary adjuvant is hyaluronidase.

In another embodiment, a pharmaceutical composition further comprises atleast one additional therapeutic agent for treating a disorder in whichIL-1β activity is detrimental.

In an embodiment, the invention provides isolated nucleic acids encodingone or more amino acid sequences of a binding protein described herein.Such nucleic acids may be inserted into a vector for carrying outvarious genetic analyses or for expressing, characterizing, or improvingone or more properties of a binding protein described herein. A vectormay comprise a one or more nucleic acid molecules encoding one or moreamino acid sequences of a binding protein described herein in which theone or more nucleic acid molecules is operably linked to appropriatetranscriptional and/or translational sequences that permit expression ofthe binding protein in a particular host cell carrying the vector.Examples of vectors for cloning or expressing nucleic acids encodingamino acid sequences of binding proteins described herein include, butare not limited, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, and pBJ.

The invention also provides a host cell comprising a vector comprising anucleic acid encoding one or more amino acid sequences of a bindingprotein described herein. Host cells useful in the invention may beprokaryotic or eukaryotic. An exemplary prokaryotic host cell isEscherichia coli. Eukaryotic cells useful as host cells in the inventioninclude protist cell, animal cell, plant cell, and fungal cell. Anexemplary fungal cell is a yeast cell, including Saccharomycescerevisiae. An exemplary animal cell useful as a host cell according tothe invention includes, but is not limited to, a mammalian cell, anavian cell, and an insect cell. Preferred mammalian cells include CHOand COS cells. An insect cell useful as a host cell according to theinvention is an insect Sf9 cell.

In another aspect, the invention provides a method of producing abinding protein described herein comprising culturing a host cellcomprising a vector encoding the binding protein in culture medium underconditions sufficient to produce the binding protein capable of bindingIL-1α and/or IL-1β. The protein so produced can be isolated and used invarious compositions and methods described herein.

In another embodiment, the invention provides a method for reducinghuman IL-1 activity comprising contacting human IL-1 with a bindingprotein described herein, such that human IL-1 activity is reduced.

Another embodiment of the invention provides a method for treating asubject for a disorder by administering to the subject a binding proteindescribed herein such that treatment is achieved.

In another embodiment, a binding protein described herein is useful fortreating a disorder selected from the group consisting of: diabetes;uveitis; neuropathic pain; osteoarthritic pain; inflammatory pain;rheumatoid arthritis; osteoarthritis; juvenile chronic arthritis; septicarthritis; Lyme arthritis; psoriatic arthritis; reactive arthritis;spondyloarthropathy; systemic lupus erythematosus (SLE); Crohn'sdisease; ulcerative colitis; inflammatory bowel disease; autoimmunediabetes; insulin dependent diabetes mellitus; thyroiditis; asthma;allergic diseases; psoriasis; dermatitis; scleroderma; graft versus hostdisease; organ transplant rejection; acute immune disease associatedwith organ transplantation; chronic immune disease associated with organtransplantation; sarcoidosis; atherosclerosis; disseminatedintravascular coagulation (DIC); Kawasaki's disease; Grave's disease;nephrotic syndrome; chronic fatigue syndrome; Wegener's granulomatosis;Henoch-Schoenlein purpurea; microscopic vasculitis of the kidneys;chronic active hepatitis; autoimmune uveitis; septic shock; toxic shocksyndrome; sepsis syndrome; cachexia; infectious diseases; parasiticdiseases; acute transverse myelitis; Huntington's chorea; Parkinson'sdisease; Alzheimer's disease; stroke; primary biliary cirrhosis;hemolytic anemia; malignancies; heart failure; myocardial infarction;Addison's disease; sporadic polyglandular deficiency type I;polyglandular deficiency type II (Schmidt's syndrome); acute respiratorydistress syndrome (ARDS); alopecia; alopecia greata; seronegativearthropathy; arthropathy; Reiter's disease; psoriatic arthropathy;ulcerative colitic arthropathy; enteropathic synovitis; chlamydia;Yersinia and Salmonella associated arthropathy; spondyloarthropathy;atheromatous disease/arteriosclerosis; atopic allergy; autoimmunebullous disease; pemphigus vulgaris; pemphigus foliaceus; pemphigoid;linear IgA disease; autoimmune haemolytic anaemia; Coombs positivehaemolytic anaemia; acquired pernicious anaemia; juvenile perniciousanaemia; myalgic encephalitis/Royal Free disease; chronic mucocutaneouscandidiasis; giant cell arteritis (GCA); primary sclerosing hepatitis;cryptogenic autoimmune hepatitis; acquired immunodeficiency syndrome(AIDS); acquired immunodeficiency related diseases; hepatitis B;hepatitis C; common varied immunodeficiency (common variablehypogammaglobulinaemia); dilated cardiomyopathy; female infertility;ovarian failure; premature ovarian failure; fibrotic lung disease;cryptogenic fibrosing alveolitis; post-inflammatory interstitial lungdisease; interstitial pneumonitis; connective tissue disease associatedinterstitial lung disease; mixed connective tissue disease associatedlung disease; systemic sclerosis associated interstitial lung disease;rheumatoid arthritis associated interstitial lung disease; systemiclupus erythematosus associated lung disease;dermatomyositis/polymyositis associated lung disease; Sjögren's diseaseassociated lung disease; ankylosing spondylitis associated lung disease;vasculitic diffuse lung disease; haemosiderosis associated lung disease;drug-induced interstitial lung disease; fibrosis; radiation fibrosis;bronchiolitis obliterans; chronic eosinophilic pneumonia; lymphocyticinfiltrative lung disease; postinfectious interstitial lung disease;gouty arthritis; autoimmune hepatitis; type-1 autoimmune hepatitis(classical autoimmune or lupoid hepatitis); type-2 autoimmune hepatitis(anti-LKM antibody hepatitis); autoimmune mediated hypoglycaemia; type Binsulin resistance with acanthosis nigricans; hypoparathyroidism;osteoarthrosis; primary sclerosing cholangitis; psoriasis type 1;psoriasis type 2; idiopathic leucopaenia; autoimmune neutropaenia; renaldisease NOS; glomerulonephritides; microscopic vasculitis of thekidneys; Lyme disease; discoid lupus erythematosus; idiopathic maleinfertility; nitric oxide-associated male infertility; spermautoimmunity; multiple sclerosis (all subtypes, including primaryprogressive, secondary progressive, relapsing remitting); sympatheticophthalmia; pulmonary hypertension secondary to connective tissuedisease; Goodpasture's syndrome; pulmonary manifestation ofpolyarteritis nodosa; acute rheumatic fever; rheumatoid spondylitis;Still's disease; systemic sclerosis; Sjörgren's syndrome; Takayasu'sdisease/arteritis; autoimmune thrombocytopaenia (AITP); idiopathicthrombocytopaenia; autoimmune thyroid disease; hyperthyroidism; goitrousautoimmune hypothyroidism (Hashimoto's disease); atrophic autoimmunehypothyroidism; primary myxoedema; phacogenic uveitis; primaryvasculitis; vitiligo; acute liver disease; chronic liver disease;alcoholic cirrhosis; alcohol-induced liver injury; cholestasis;idiosyncratic liver disease; drug-induced hepatitis; non-alcoholicsteatohepatitis; allergy; group B Streptococci (GBS) infection; mentaldisorders (e.g., depression and schizophrenia); Th2 Type and Th1 Typemediated diseases; acute and chronic pain (different forms of pain);cancer (such as lung, breast, stomach, bladder, colon, pancreas,ovarian, prostate, and rectal cancer); hematopoietic malignancies;leukemia; lymphoma; abetalipoproteinemia; acrocyanosis; acute andchronic parasitic or infectious processes; acute leukemia; acutelymphoblastic leukemia (ALL); T-cell ALL; FAB ALL; acute myeloidleukemia (AML); acute or chronic bacterial infection; acutepancreatitis; acute renal failure; adenocarcinomas; atrial ectopicbeats; AIDS dementia complex; alcohol-induced hepatitis; allergicconjunctivitis; allergic contact dermatitis; allergic rhinitis;allograft rejection; alpha-1-antitrypsin deficiency; amyotrophic lateralsclerosis; anemia; angina pectoris; anterior horn cell degeneration;anti-CD3 therapy; antiphospholipid syndrome; anti-receptorhypersensitivity reactions; aortic and peripheral aneurysms; aorticdissection; arterial hypertension; arteriosclerosis; arteriovenousfistula; ataxia; atrial fibrillation (sustained or paroxysmal); atrialflutter; atrioventricular block; B cell lymphoma; bone graft rejection;bone marrow transplant (BMT) rejection; bundle branch block; Burkitt'slymphoma; burns; cardiac arrhythmias; cardiac stun syndrome; cardiactumors; cardiomyopathy; cardiopulmonary bypass inflammation response;cartilage transplant rejection; cerebellar cortical degenerations;cerebellar disorders; chaotic or multifocal atrial tachycardia;chemotherapy associated disorders; chronic myelocytic leukemia (CML);chronic alcoholism; chronic inflammatory pathologies; chroniclymphocytic leukemia (CLL); chronic obstructive pulmonary disease(COPD); chronic salicylate intoxication; colorectal carcinoma;congestive heart failure; conjunctivitis; contact dermatitis; corpulmonale; coronary artery disease; Creutzfeldt-Jakob disease; culturenegative sepsis; cystic fibrosis; cytokine therapy associated disorders;dementia pugilistica; demyelinating diseases; dengue hemorrhagic fever;dermatitis; dermatologic conditions; diabetes mellitus; diabeticarteriosclerotic disease; diffuse Lewy body disease; dilated congestivecardiomyopathy; disorders of the basal ganglia; Down's syndrome inmiddle age; drug-induced movement disorders induced by drugs which blockCNS dopamine receptors; drug sensitivity; eczema; encephalomyelitis;endocarditis; endocrinopathy; epiglottitis; Epstein-Barr virusinfection; erythromelalgia; extrapyramidal and cerebellar disorders;familial hemophagocytic lymphohistiocytosis; fetal thymus implantrejection; Friedreich's ataxia; functional peripheral arterialdisorders; fungal sepsis; gas gangrene; gastric ulcer; glomerularnephritis; graft rejection of any organ or tissue; gram negative sepsis;gram positive sepsis; granulomas due to intracellular organisms; hairycell leukemia; Hallervorden-Spatz disease; Hashimoto's thyroiditis; hayfever; heart transplant rejection; hemochromatosis; hemodialysis;hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura;hemorrhage; hepatitis A; His bundle arrhythmias; HIV infection/HIVneuropathy; Hodgkin's disease; hyperkinetic movement disorders;hypersensitivity reactions; hypersensitivity pneumonitis; hypertension;hypokinetic movement disorders; hypothalamic-pituitary-adrenal axisevaluation; idiopathic Addison's disease; idiopathic pulmonary fibrosis(IPF); antibody mediated cytotoxicity; asthenia; infantile spinalmuscular atrophy; inflammation of the aorta; influenza a; ionizingradiation exposure; iridocyclitis/uveitis/optic neuritis;ischemia-reperfusion injury; ischemic stroke; juvenile rheumatoidarthritis; juvenile spinal muscular atrophy; Kaposi's sarcoma; kidneytransplant rejection; legionella; leishmaniasis; leprosy; lesions of thecorticospinal system; lipedema; liver transplant rejection; lymphedema;malaria; malignant lymphoma; malignant histiocytosis; malignantmelanoma; meningitis; meningococcemia; metabolic syndrome migraineheadache; idiopathic migraine headache; mitochondrial multisystemdisorder; mixed connective tissue disease; monoclonal gammopathy;multiple myeloma; multiple systems degenerations (Menzel;Dejerine-Thomas; Shy-Drager; and Machado-Joseph); myasthenia gravis;mycobacterium avium intracellulare; mycobacterium tuberculosis;myelodysplastic syndrome; myocardial infarction; myocardial ischemicdisorders; nasopharyngeal carcinoma; neonatal chronic lung disease;nephritis; nephrosis; neurodegenerative diseases; neurogenic I muscularatrophies; neutropenic fever; non-Hodgkin's lymphoma; occlusion of theabdominal aorta and its branches; occlusive arterial disorders; OKT3®therapy; orchitis/epididymitis; orchitis/vasectomy reversal procedures;organomegaly; osteoporosis; pancreas transplant rejection; pancreaticcarcinoma; paraneoplastic syndrome/hypercalcemia of malignancy;parathyroid transplant rejection; pelvic inflammatory disease; perennialrhinitis; pericardial disease; peripheral atherosclerotic disease;peripheral vascular disorders; peritonitis; pernicious anemia;pneumocystis carinii pneumonia; pneumonia; POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome); post perfusion syndrome; post pump syndrome;post-MI cardiotomy syndrome; preeclampsia; progressive supranucleopalsy; primary pulmonary hypertension; radiation therapy; Raynaud'sphenomenon; Raynaud's disease; Refsum's disease; regular narrow QRStachycardia; renovascular hypertension; reperfusion injury; restrictivecardiomyopathy; sarcomas; senile chorea; senile dementia of Lewy bodytype; seronegative arthropathies; shock; sickle cell anemia; skinallograft rejection; skin changes syndrome; small bowel transplantrejection; solid tumors; specific arrhythmias; spinal ataxia;spinocerebellar degenerations; streptococcal myositis; structurallesions of the cerebellum; subacute sclerosing panencephalitis; syncope;syphilis of the cardiovascular system; systemic anaphylaxis; systemicinflammatory response syndrome; systemic onset juvenile rheumatoidarthritis; telangiectasia; thromboangitis obliterans; thrombocytopenia;toxicity; transplants; trauma/hemorrhage; type III hypersensitivityreactions; type IV hypersensitivity; unstable angina; uremia; urosepsis;urticaria; valvular heart diseases; varicose veins; vasculitis; venousdiseases; venous thrombosis; ventricular fibrillation; viral and fungalinfections; viral encephalitis/aseptic meningitis; viral-associatedhemophagocytic syndrome; Wernicke-Korsakoff syndrome; Wilson's disease;xenograft rejection of any organ or tissue; acute coronary syndromes;acute idiopathic polyneuritis; acute inflammatory demyelinatingpolyradiculoneuropathy; acute ischemia; adult Still's disease; alopeciagreata; anaphylaxis; anti-phospholipid antibody syndrome; aplasticanemia; arteriosclerosis; atopic eczema; atopic dermatitis; autoimmunedermatitis; autoimmune disorder associated with Streptococcus infection;autoimmune enteropathy; autoimmune hearing loss; autoimmunelymphoproliferative syndrome (ALPS); autoimmune myocarditis; autoimmunepremature ovarian failure; blepharitis; bronchiectasis; bullouspemphigoid; cardiovascular disease; catastrophic antiphospholipidsyndrome; celiac disease; cervical spondylosis; chronic ischemia;cicatricial pemphigoid; clinically isolated syndrome (CIS) with risk formultiple sclerosis; conjunctivitis; childhood onset psychiatricdisorder; dacryocystitis; dermatomyositis; diabetic retinopathy; diskherniation; disk prolapse; drug induced immune hemolytic anemia;endocarditis; endometriosis; endophthalmitis; episcleritis; erythemamultiforme; erythema multiforme major; gestational pemphigoid;Guillain-Barré syndrome (GBS); hay fever; Hughes syndrome; idiopathicParkinson's disease; idiopathic interstitial pneumonia; IgE-mediatedallergy; immune hemolytic anemia; inclusion body myositis; infectiousocular inflammatory disease; inflammatory demyelinating disease;inflammatory heart disease; inflammatory kidney disease; iritis;keratitis; keratojunctivitis sicca; Kussmaul disease or Kussmaul-Meierdisease; Landry's paralysis; Langerhan's cell histiocytosis; livedoreticularis; macular degeneration; microscopic polyangiitis; MorbusBechterev; motor neuron disorders; mucous membrane pemphigoid; multipleorgan failure; myasthenia gravis; myelodysplastic syndrome; myocarditis;nerve root disorders; neuropathy; non-A non-B hepatitis; optic neuritis;osteolysis; pauciarticular JRA; peripheral artery occlusive disease(PAOD); peripheral vascular disease (PVD); peripheral artery; disease(PAD); phlebitis; polyarteritis nodosa (or periarteritis nodosa);polychondritis; polymyalgia rheumatica; poliosis; polyarticular JRA;polyendocrine deficiency syndrome; polymyositis; polymyalgia rheumatica(PMR); post-pump syndrome; primary Parkinsonism; secondary Parkinsonism;prostatitis; pure red cell aplasia; primary adrenal insufficiency;recurrent neuromyelitis optica; restenosis; rheumatic heart disease;SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis);secondary amyloidosis; shock lung; scleritis; sciatica; secondaryadrenal insufficiency; silicone associated connective tissue disease;Sneddon-Wilkinson dermatosis; spondylitis ankylosans; Stevens-Johnsonsyndrome (SJS); systemic inflammatory response syndrome; temporalarteritis; toxoplasmic retinitis; toxic epidermal necrolysis; transversemyelitis; TRAPS (tumor necrosis factor receptor type 1 (TNFR)-associatedperiodic syndrome); type B insulin resistance with acanthosis nigricans;type 1 allergic reaction; type II diabetes; urticaria; usualinterstitial pneumonia (UIP); vernal conjunctivitis; viral retinitis;Vogt-Koyanagi-Harada syndrome (VKH syndrome); wet macular degeneration;wound healing; Yersinia and Salmonella associated arthropathy.

In a further embodiment of a method of treatment described herein, thestep of administering to the subject a binding protein or bindng proteinconstruct or binding protein conjugate described herein is by at leastone mode selected from parenteral, subcutaneous, intramuscular,intravenous, intra-articular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

Another aspect of the invention is a method of treating a patientsuffering from a disorder in which IL-1 is detrimental comprising thestep of administering a binding protein, binding protein construct, orbinding protein conjugate described herein before, concurrently with, orafter the administration of a second agent, wherein the second agent isselected from the group consisting of inhaled steroids; beta-agonists;short-acting or long-acting beta-agonists; antagonists of leukotrienesor leukotriene receptors; ADVAIR; IgE inhibitors; anti-IgE antibodies;XOLAIR; phosphodiesterase inhibitors; PDE4 inhibitors; xanthines;anticholinergic drugs; mast cell-stabilizing agents; Cromolyn; IL-4inhibitors; IL-5 inhibitors; eotaxin/CCR3 inhibitors; antagonists ofhistamine or its receptors including H1, H2, H3, and H4; antagonists ofprostaglandin D or its receptors DP1 and CRTH2; TNF antagonists; asoluble fragment of a TNF receptor; ENBREL®; TNF enzyme antagonists; TNFconverting enzyme (TACE) inhibitors; muscarinic receptor antagonists;TGF-beta antagonists; interferon gamma; perfenidone; chemotherapeuticagents, methotrexate; leflunomide; sirolimus (rapamycin) or an analogthereof, CCI-779; COX2 or cPLA2 inhibitors; NSAIDs; immunomodulators;p38 inhibitors; TPL-2, MK-2 and NFkB inhibitors; budenoside; epidermalgrowth factor; corticosteroids; cyclosporine; sulfasalazine;aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole;lipoxygenase inhibitors; mesalamine; olsalazine; balsalazide;antioxidants; thromboxane inhibitors; IL-1 receptor antagonists;anti-IL-11 antibodies; anti-IL-6 antibodies; growth factors; elastaseinhibitors; pyridinyl-imidazole compounds; antibodies or agonists ofTNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21,IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31,IL-32, IL-33, EMAP-II, GM-CSF, FGF, or PDGF; antibodies of CD2, CD3,CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD90 or their ligands;FK506; rapamycin; mycophenolate mofetil; ibuprofen; prednisolone;phosphodiesterase inhibitors; adensosine agonists; antithromboticagents; complement inhibitors; adrenergic agents; IRAK, NIK, IKK, p38,or MAP kinase inhibitors; IL-1β converting enzyme inhibitors; TNF-αconverting enzyme inhibitors; T-cell signaling inhibitors;metalloproteinase inhibitors; 6-mercaptopurines; angiotensin convertingenzyme inhibitors; soluble cytokine receptors; soluble p55 TNF receptor;soluble p75 TNF receptor; sIL-1RI; sIL-1RII; sIL-6R; anti-inflammatorycytokines; IL-4; IL-10; IL-11; and TGF-β.

Another aspect of the invention provides at least one IL-1 anti-idiotypeantibody to at least one IL-1 binding protein described herein. Theanti-idiotype antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulin moleculesuch as, but not limited to, at least one CDR of a heavy or light chainor ligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that can be incorporated into a bindingprotein of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to IL-1β binding proteins, including, but notlimited to, anti-IL-1β antibodies, or antigen-binding portions thereof,that bind IL-1β and multivalent, multispecific binding proteins such asDVD-Ig™ that bind IL-1β and another target. Various aspects of theinvention relate to antibodies and antibody fragments, DVD-Ig bindingproteins, and pharmaceutical compositions thereof, as well as nucleicacids, recombinant expression vectors and host cells for making suchIL-1β binding proteins, including antibodies, DVD-Ig binding proteins,and fragments thereof. Methods of using the IL-113 binding proteins ofthe invention to detect human IL-1β; to inhibit human IL-1β, either invitro or in vivo; and to regulate gene expression are also encompassedby the invention.

The invention also encompasses any binding protein or antibody capableof competing with an IL-1β binding protein described herein.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present invention may be more readily understood, select termsare defined below.

The term “polypeptide” refers to any polymeric chain of amino acids. Theterms “peptide” and “protein” are used interchangeably with the termpolypeptide and also refer to a polymeric chain of amino acids. The term“polypeptide” encompasses native or artificial proteins, proteinfragments and polypeptide analogs of a protein sequence. A polypeptidemay be monomeric or polymeric The term “polypeptide” encompassesfragments and variants (including fragments of variants) thereof, unlessotherwise contradicted by context. For an antigenic polypeptide, afragment of polypeptide optionally contains at least one contiguous ornonlinear epitope of polypeptide. The precise boundaries of the at leastone epitope fragment can be confirmed using ordinary skill in the art.The fragment comprises at least about 5 contiguous amino acids, such asat least about 10 contiguous amino acids, at least about 15 contiguousamino acids, or at least about 20 contiguous amino acids. A variant ofpolypeptide is as described herein.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” refers to the process of rendering a chemicalspecies such as a polypeptide substantially free of naturally associatedcomponents by isolation, e.g., using protein purification techniqueswell known in the art.

The term “human IL-1α” (abbreviated herein as hIL-1a, or IL-1α),includes a pleiotropic cytokine involved in various immune responses,inflammatory processes, and hematopoiesis. For example, IL-1α includesthe human cytokine produced by activated macrophages; it stimulatesthymocyte proliferation by inducing IL-2 release, B-cell maturation andproliferation, and fibroblast growth factor activity. The term humanIL-1α is intended to include recombinant human IL-1α (rh IL-1α) that canbe prepared by standard recombinant expression methods.

The term “human IL-1β” (abbreviated herein as hIL-1β, or IL-1β) includesa pleiotropic cytokine involved in various immune responses,inflammatory processes, and hematopoiesis. The term human IL-1β includesrecombinant human IL-1β (rh IL-1β) that can be prepared by standardrecombinant expression methods.

The amino acid sequences of human IL-1α and IL-1β are shown in Table 1.

TABLE 1 Sequence of Human IL-1α and Human IL-1β Sequence SequenceProtein Identifier 123456789012345678901234567890 Human SEQ ID NO:SAPFSFLSNVKYNFMRIIKYEFILNDALNQ mature 1 SIIRANDQYLTAAALHNLDEAVKFDMGAYKIL-1α SSKDDAKITVILRISKTQLYVTAQDEDQPV LLKEMPEIPKTITGSETNLLFFWETHGTKNYFTSVAHPNLFIATKQDYWVCLAGGPPSIT DFQILENQA Human SEQ ID NO:APVRSLNCTLRDSQQKSLVMSGPYELKALH mature 2 LQGQDMEQQVVFSMSFVQGEESNDKIPVALIL-1β GLKEKNLYLSCVLKDDKPTLQLESVDPKNY PKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQF VSS

The term “biological activity” refers to all inherent biologicalproperties of the IL-1 cytokine, e.g., IL-1α and/or IL-1β. Biologicalproperties of IL-1α and IL-1β include, but are not limited to, bindingto an IL-1 receptor.

The terms “specific binding” or “specifically binding” in reference tothe interaction of an antibody, a protein, or a peptide with a secondchemical species, mean that the interaction is dependent upon thepresence of a particular structure (e.g., an antigenic determinant orepitope) on the chemical species; for example, an antibody recognizesand binds to a specific protein structure rather than to proteinsgenerally. If an antibody is specific for epitope “A”, the presence of amolecule containing epitope A (or free, unlabeled A), in a reactioncontaining labeled “A” and the antibody, will reduce the amount oflabeled A bound to the antibody.

The term “antibody” broadly refers to any immunoglobulin (Ig) moleculecomprised of four polypeptide chains, two heavy (H) chains and two light(L) chains, or any functional fragment, mutant, variant, or derivationthereof, which retains the essential epitope binding features of an Igmolecule. Such mutant, variant, or derivative antibody formats are knownin the art. Nonlimiting embodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains: CH1, CH2, and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 andIgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain, and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (Winter et al., U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fcportion of an antibody mediates several important effector functions,for example, cytokine induction, ADCC, phagocytosis, complementdependent cytotoxicity (CDC), and half-life/clearance rate of antibodyand antigen-antibody complexes. In some cases these effector functionsare desirable for therapeutic antibody but in other cases might beunnecessary or even deleterious, depending on the therapeuticobjectives. Certain human IgG isotypes, particularly IgG1 and IgG3,mediate ADCC and CDC via binding to FcγRs and complement Clq,respectively. Neonatal Fc receptors (FcRn) are the critical componentsdetermining the circulating half-life of antibodies. In still anotherembodiment at least one amino acid residue is replaced in the constantregion of the antibody, for example the Fc region of the antibody, suchthat effector functions of the antibody are altered. The dimerization oftwo identical heavy chains of an immunoglobulin is mediated by thedimerization of CH3 domains and is stabilized by the disulfide bondswithin the hinge region (Huber et al., Nature, 264: 415-420 (1976);Thies et al., J. Mol. Biol., 293: 67-79 (1999)). Mutation of cysteineresidues within the hinge regions to prevent heavy chain-heavy chaindisulfide bonds will destabilize dimerization of CH3 domains. Residuesresponsible for CH3 dimerization have been identified (Dall′ Acqua etal., Biochemistry, 37: 9266-9273 (1998)). Therefore, it is possible togenerate a monovalent half-Ig. Interestingly, these monovalent half Igmolecules have been found in nature for both IgG and IgA subclasses(Seligmann et al., Ann. Immunol., 129 C: 855-870 (1978); Biewenga etal., Clin. Exp. Immunol., 51: 395-400 (1983)). The stoichiometry ofFcRn: Ig Fc region has been determined to be 2:1 (West et al.,Biochemistry, 39: 9698-9708 (2000)), and half Fc is sufficient formediating FcRn binding (Kim et al., Eur. J. Immunol., 24: 542-548(1994)). Mutations to disrupt the dimerization of CH3 domain may nothave greater adverse effect on its FcRn binding as the residuesimportant for CH3 dimerization are located on the inner interface of CH3b sheet structure, whereas the region responsible for FcRn binding islocated on the outside interface of CH2-CH3 domains. However, the halfIg molecule may have certain advantage in tissue penetration due to itssmaller size than that of a regular antibody. In one embodiment, atleast one amino acid residue is replaced in the constant region of thebinding protein of the invention, for example the Fc region, such thatthe dimerization of the heavy chains is disrupted, resulting in half DVDIg molecules. The anti-inflammatory activity of IgG is completelydependent on sialylation of the N-linked glycan of the IgG Fc fragment.The precise glycan requirements for anti-inflammatory activity has beendetermined, such that an appropriate IgG1 Fc fragment can be created,thereby generating a fully recombinant, sialylated IgG1 Fc with greatlyenhanced potency (Anthony et al., Science, 320:373-376 (2008)).

The term “antigen-binding portion” of an antibody refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., hIL-1β). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Such antibody embodiments may also be bispecific, dualspecific, or multi-specific formats; specifically binding to two or moredifferent antigens (e.g., hIL-1β and a different antigen molecule, suchas hIL-1β and hIL-1α). Examples of binding fragments encompassed withinthe term “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL, and CH1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody, (v)a dAb fragment (Ward et al., Nature, 341: 544-546 (1989); PCTPublication No. WO 90/05144), which comprises a single variable domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see, for example, Bird etal., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad.Sci. USA, 85: 5879-5883 (1988)). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Other forms of single chain antibodies, such as diabodiesare also encompassed. Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA,90: 6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)).Such antibody binding portions are known in the art (Kontermann andDiibel eds., Antibody Engineering (Springer-Verlag, New York, 2001), p.790 (ISBN 3-540-41354-5)). In addition single chain antibodies alsoinclude “linear antibodies” comprising a pair of tandem Fv segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.,Protein Eng., 8(10): 1057-1062 (1995); and U.S. Pat. No. 5,641,870)).

An immunoglobulin constant (C) domain refers to a heavy (CH) or light(CL) chain constant domain. Murine and human IgG heavy chain and lightchain constant domain amino acid sequences are known in the art.

The term “IL-1β binding protein construct” (or “binding proteinconstruct”) refers to a polypeptide comprising one or more of theantigen binding portions of the invention linked to a linker or animmunoglobulin constant domain. A “linker polypeptide” comprises two ormore amino acid residues joined by peptide bonds and are used to linkone or more antigen binding portions. Such linker polypeptides are wellknown in the art (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA,90: 6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)). Animmunoglobulin constant domain refers to a heavy or light chain constantdomain. Human IgG heavy chain and light chain constant domain amino acidsequences are known in the art and represented in Table 2.

TABLE 2 Sequence of Human IgG Heavy Chain Constant Domainand Light Chain Constant Domain Sequence Identi- Sequence Protein fier123456789012345678901234567890 Ig gamma-1 SEQ IDASTKGPSVFFLAPSSKSTSGGTAALGCLVK constant NO: 3DYFPEPVTVSWNSGALTSGVHTFPAVLQSS region GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig gamma-1SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALGCLVK constant NO: 4DYFPEPVTVSWNSGALTSGVHTFPAVLQSS region GLYSLSSVVTVPSSSLGTQTYICNVNHKPSmutant NTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig KappaSEQ ID TVAAPSVFIFPPSDEQLKSGTASVVCLLNN constant NO: 5FYPREAKVQWKVDNALQSGNSQESVTEQDS region KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ig Lambda SEQ ID QPKAAPSVTLFPPSSEELQANKATLVCLISconstant NO: 6 DFYPGAVTVAWKADSSPVKAGVETTTPSKQ regionSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS

Still further, an IL-1β binding protein, such as an antibody orantigen-binding portion thereof, may be part of a larger immunoadhesionmolecule, formed by covalent or noncovalent association of the antibodyor antigen-binding portion with one or more other proteins or peptides.Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanovet al., Human Antibod. Hybridomas, 6: 93-101 (1995)) and use of acysteine residue, a marker peptide and a C-terminal polyhistidine tag tomake bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol.Immunol., 31: 1047-1058 (1994)). Antibody portions, such as Fab andF(ab′)₂ fragments, can be prepared from whole antibodies usingconventional techniques, such as papain or pepsin digestion,respectively, of whole antibodies. Moreover, antibodies, antigen-bindingportions thereof, and immunoadhesion molecules can be obtained usingstandard recombinant DNA techniques.

An “isolated antibody” is intended to refer to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds hIL-1βis substantially free of antibodies that specifically bind antigensother than hIL-1β). An isolated antibody that specifically binds hIL-1βmay, however, have cross-reactivity to other antigens, such as IL-1βmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The term “monoclonal antibody” or “mAb” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each mAb is directedagainst a single determinant on the antigen. The modifier “monoclonal”is not to be construed as requiring production of the antibody by anyparticular method.

The term “human antibody” includes antibodies having variable andconstant regions derived from human germline immunoglobulin sequences.The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs and in particular CDR3.However, the term “human antibody” does not include antibodies in whichCDR sequences derived from the germline of another mammalian species,such as a mouse, have been grafted onto human framework sequences.

The term “recombinant human antibody” includes all human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell (described further in Section II C, below),antibodies isolated from a recombinant, combinatorial human antibodylibrary (Hoogenboom, H. R., Trends Biotechnol., 15: 62-70 (1997); Azzazyand Highsmith, Clin. Biochem., 35: 425-445 (2002); Gavilondo andLarrick, BioTechniques, 29: 128-145 (2000); Hoogenboom and Chames,Immunol. Today, 21: 371-378 (2000)), antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (see,e.g., Taylor et al., Nucl. Acids Res., 20: 6287-6295 (1992); Kellermannand Green, Curr. Opin. Biotechnol., 13: 593-597 (2002); Little et al.,Immunol. Today, 21: 364-370 (2000)); or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies that comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies that comprise heavyand light chain variable region sequences from one species but in whichthe sequences of one or more of the CDR regions of VH and/or VL arereplaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “CDR” refers to the complementarity determining region withinantibody variable sequences. There are three CDRs in each of thevariable regions of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable regions. Theterm “CDR set” as used herein refers to a group of three CDRs that occurin a single variable region capable of binding the antigen. The exactboundaries of these CDRs have been defined differently according todifferent systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987); andChothia et al., Nature, 342: 877-883 (1989)) found that certainsub-portions within Kabat CDRs adopt nearly identical peptide backboneconformations, despite having great diversity at the level of amino acidsequence. These sub-portions were designated as L1, L2, and L3 or H1,H2, and H3 where the “L” and the “H” designates the light chain and theheavy chains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan et al. (FASEB J., 9: 133-139 (1995)) and MacCallumet al. (J. Mol. Biol., 262(5): 732-745 (1996)). Still other CDR boundarydefinitions may not strictly follow one of the above systems, but willnonetheless overlap with the Kabat CDRs, although they may be shortenedor lengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughexemplary embodiments use Kabat or Chothia defined CDRs.

The terms “Kabat numbering”, “Kabat definitions”, and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al., Ann. NY Acad. Sci., 190: 382-391(1971); and Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 (1991)). For the heavy chain variableregion, the hypervariable region ranges from amino acid positions 31 to35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acidpositions 95 to 102 for CDR3. For the light chain variable region, thehypervariable region ranges from amino acid positions 24 to 34 for CDR1,amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to97 for CDR3.

The growth and analysis of extensive public databases of amino acidsequences of variable heavy and light regions over the past twenty yearshave led to the understanding of the typical boundaries betweenframework regions (FR) and CDR sequences within variable regionsequences and enabled persons skilled in this art to accuratelydetermine the CDRs according to Kabat numbering, Chothia numbering, orother systems. See, e.g., Martin, “Protein Sequence and StructureAnalysis of Antibody Variable Domains,” Chapter 31, In AntibodyEngineering, (Kontermann and Diibel, eds.) (Springer-Verlag, Berlin,2001), especially pages 432-433. A useful method of determining theamino acid sequences of Kabat CDRs within the amino acid sequences ofvariable heavy (VH) and variable light (VL) regions is provided below:

To identify a CDR-L1 amino acid sequence:

-   -   Starts approximately 24 amino acid residues from the amino        terminus of the VL region;    -   Residue before the CDR-L1 sequence is always cysteine (C);    -   Residue after the CDR-L1 sequence is always a tryptophan (W)        residue,    -   typically Trp-Tyr-Gln (W-Y-Q), but also Trp-Leu-Gln (W-L-Q),        Trp-Phe-Gln    -   (W-F-Q), and Trp-Tyr-Leu (W-Y-L);    -   Length is typically 10 to 17 amino acid residues.

To identify a CDR-L2 amino acid sequence:

-   -   Starts always 16 residues after the end of CDR-L1;    -   Residues before the CDR-L2 sequence are generally Ile-Tyr (1-Y),        but also Val-Tyr (V-Y), Ile-Lys (I-K), and Ile-Phe (I-F);    -   Length is always 7 amino acid residues.

To identify a CDR-L3 amino acid sequence:

-   -   Starts always 33 amino acids after the end of CDR-L2;    -   Residue before the CDR-L3 amino acid sequence is always a        cysteine (C);    -   Residues after the CDR-L3 sequence are always Phe-Gly-X-Gly        (F-G-X-G) (SEQ ID NO:11), where X is any amino acid;    -   Length is typically 7 to 11 amino acid residues.

To identify a CDR-H1 amino acid sequence:

-   -   Starts approximately 31 amino acid residues from amino terminus        of VH region and always 9 residues after a cysteine (C);    -   Residues before the CDR-H1 sequence are always        Cys-X-X-X-X-X-X-X-X (SEQ ID NO:12), where X is any amino acid;    -   Residue after CDR-H1 sequence is always a Trp (W), typically        Trp-Val (W-V), but also Trp-Ile (W-I), and Trp-Ala (W-A);    -   Length is typically 5 to 7 amino acid residues.

To identify a CDR-H2 amino acid sequence:

-   -   Starts always 15 amino acid residues after the end of CDR-H1;    -   Residues before CDR-H2 sequence are typically        Leu-Glu-Trp-Ile-Gly (L-E-W-I-G) (SEQ ID NO:23), but other        variations also;    -   Residues after CDR-H2 sequence are        Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala        (K/R-L/I/V/F/T/A-T/S/I/A);    -   Length is typically 16 to 19 amino acid residues.

To identify a CDR-H3 amino acid sequence:

-   -   Starts always 33 amino acid residues after the end of CDR-H2 and        always 3 after a cysteine (C)′    -   Residues before the CDR-H3 sequence are always Cys-X-X (C-X-X),        where X is any amino acid, typically Cys-Ala-Arg (C-A-R);    -   Residues after the CDR-H3 sequence are always Trp-Gly-X-Gly        (W-G-X-G) (SEQ ID NO:24), where X is any amino acid;    -   Length is typically 3 to 25 amino acid residues.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol., 196: 901-917 (1987)); andChothia et al. (J. Mol. Biol., 227: 799-817 (1992)), both areincorporated herein by reference). According to Chothia et al., criticalportions of the CDRs of many antibodies have nearly identical peptidebackbone confirmations despite great diversity at the level of aminoacid sequence. Each canonical structure specifies primarily a set ofpeptide backbone torsion angles for a contiguous segment of amino acidresidues forming a loop.

An “affinity matured” antibody is an antibody with one or morealterations in one or more CDRs thereof which result in an improvementin the affinity of the antibody for a target antigen, compared to aparent antibody which does not possess the alteration(s). Exemplaryaffinity matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. A variety of procedures for producingaffinity matured antibodies are known in the art. For example, Marks etal., BioTechnology, 10: 779-783 (1992) describes affinity maturation byVH and VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); Hawkins et al., J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

The term “multivalent binding protein” denotes a binding proteincomprising two or more antigen binding sites. A multivalent bindingprotein is preferably engineered to have three or more antigen bindingsites, and is generally not a naturally occurring antibody. The term“multispecific binding protein” refers to a binding protein capable ofbinding two or more related or unrelated targets. “Dual variable domain”(“DVD”) binding proteins of the invention comprise two or more antigenbinding sites and are tetravalent or multivalent binding proteins. DVDsmay be monospecific, i.e., capable of binding one antigen, ormultispecific, i.e., capable of binding two or more antigens. A DVDbinding protein comprising two heavy chain DVD polypeptides and twolight chain DVD polypeptides is referred to as a “DVD immunoglobulin” or“DVD-Ig”. Each half of a DVD-Ig comprises a heavy chain DVD polypeptideand a light chain DVD polypeptide, and two or more antigen bindingsites. Each binding site comprises a heavy chain variable domain and alight chain variable domain with a total of six CDRs involved in antigenbinding per antigen binding site.

A description of the design, expression, and characterization of DVD-Igmolecules is provided in PCT Publication No. WO 2007/024715; U.S. Pat.No. 7,612,181; and Wu et al., Nature Biotechnol., 25: 1290-1297 (2007).A preferred example of such DVD-Ig molecules comprises a heavy chainthat comprises the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1is a first heavy chain variable domain, VD2 is a second heavy chainvariable domain, C is a heavy chain constant domain, X1 is a linker withthe proviso that it is not CH₁, X2 is an Fc region, and n is 0 or 1, butpreferably 1; and a light chain that comprises the structural formulaVD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variabledomain, VD2 is a second light chain variable domain, C is a light chainconstant domain, X1 is a linker with the proviso that it is not CH1, andX2 does not comprise an Fc region; and n is 0 or 1, but preferably 1.Such a DVD-Ig may comprise two such heavy chains and two such lightchains, wherein each chain comprises variable domains linked in tandemwithout an intervening constant region between variable regions, whereina heavy chain and a light chain associate to form tandem functionalantigen binding sites, and a pair of heavy and light chains mayassociate with another pair of heavy and light chains to form atetrameric binding protein with four functional antigen binding sites.In another example, a DVD-Ig molecule may comprise heavy and lightchains that each comprise three variable domains (VD1, VD2, VD3) linkedin tandem without an intervening constant region between variabledomains, wherein a pair of heavy and light chains may associate to formthree antigen binding sites, and wherein a pair of heavy and lightchains may associate with another pair of heavy and light chains to forma tetrameric binding protein with six antigen binding sites.

A DVD-Ig binding protein may bind one or more epitopes of IL-1β. ADVD-Ig binding protein may also bind an epitope of IL-1β and an epitopeof a second target antigen other than an IL-1β polypeptide.

The term “bispecific antibody”, as used herein, refers to full-lengthantibodies that are generated by quadroma technology (see Milstein andCuello, Nature, 305: 537-540 (1983)), by chemical conjugation of twodifferent monoclonal antibodies (see Staerz et al., Nature, 314: 628-631(1985)), or by knob-into-hole or similar approaches which introducesmutations in the Fc region (see Holliger et al., Proc. Natl. Acad. Sci.USA, 90(14): 6444-6448 (1993)), resulting in multiple differentimmunoglobulin species of which only one is the functional bispecificantibody. By molecular function, a bispecific antibody binds one antigen(or epitope) on one of its two binding arms (one pair of HC/LC), andbinds a different antigen (or epitope) on its second arm (a differentpair of HC/LC). By this definition, a bispecific antibody has twodistinct antigen binding arms (in both specificity and CDR sequences),and is monovalent for each antigen it binds.

The term “dual-specific antibody”, as used herein, refers to full-lengthantibodies that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT Publication No. WO02/02773). Accordingly a dual-specific binding protein has two identicalantigen binding arms, with identical specificity and identical CDRsequences, and is bivalent for each antigen to which it binds.

A “functional antigen binding site” of a binding protein is one that iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

The term “cytokine” is a generic term for proteins that are released byone cell population and that act on another cell population asintercellular mediators. Examples of such cytokines are lymphokines,monokines, and traditional polypeptide hormones. Included among thecytokines are growth hormones, such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones, such as follicle stimulating hormone (FSH), thyroidstimulating hormone (TSH), and luteinizing hormone (LH); hepatic growthfactor; fibroblast growth factor; prolactin; placental lactogen; a tumornecrosis factor such as tumor necrosis factor-alpha (TNF-α) and tumornecrosis factor-beta (TNF-β); mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-alpha (NGF-α); platelet-growth factor; placental growth factor;transforming growth factors (TGFs) such as TGF-alpha (TGF-α) andTGF-beta (TGF-β) insulin-like growth factor-1 and -11; erythropoietin(EPO); osteoinductive factors; interferons such as interferon-alpha(IFN-α), interferon-beta (IFN-β), and interferon-gamma (IFN-γ); colonystimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocytemacrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs)such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-33;and other polypeptide factors including LIF and kit ligand (KL). As usedherein, the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In an exemplary embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, —H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody providing or nucleic acid sequence encoding at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. In someembodiments, the term “acceptor” refers to the antibody amino acidproviding or nucleic acid sequence encoding the constant region(s). Inyet another embodiment, the term “acceptor” refers to the antibody aminoacid providing or nucleic acid sequence encoding one or more of theframework regions and the constant region(s). In a specific embodiment,the term “acceptor” refers to a human antibody amino acid or nucleicacid sequence that provides or encodes at least 80%, preferably, atleast 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell known in the art, antibodies in development, or antibodiescommercially available).

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the invention the human heavy chain and lightchain acceptor sequences are selected from the sequences listed fromV-base (http://vbase.mrc-cpe.cam.ac.uk/) or from IMGT®, theinternational ImMunoGeneTics information System®(http://imgt.cines.fr/textes/IMGTrepertoire/LocusGenes/). In anotherembodiment of the invention the human heavy chain and light chainacceptor sequences are selected from the sequences described in Table 3and Table 4.

TABLE 3 Heavy Chain Acceptor Sequences SEQ ID Sequence NO:Protein region 123456789012345678901234567890 7 FR1EVQLVESGGGVVQPGRSLRLSCSSSGFIFS 8 FR2 WVRQAPGKGLEWVA 9 FR3RFTISRDNSKNTLFLQMDSLRPEDTGVYFC AR 10 FR4 WGQGTPVTVSS 240 VH3-7 FR1EVQLVESGGGLVQPGGSLRLSCAASGFTFS 241 VH3-7 FR2 WVRQAPGKGLEWVA 242VH3-7 FR3 RFTISRDNAKNSLYLQMNSLRAEDTAVYYC AR 243 JH4 FR4 WGQGTLVTVSS 244VH3 CONSENSUS EVQLVESGGGLVQPGGSLRLSCAASGFTFS FR1 245 VH3 CONSENSUSWVRQAPGKGLEWVS FR2 246 VH3 CONSENSUS RFTISRDNSKNTLYLQMNSLRAEDTAVYYC FR3AR 247 JH4 FR4 WGQGTLVTVSS 248 VH1-46 FR1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT249 VH1-46 FR2 WVRQAPGQGLEWMG 250 VH1-46 FR3RVTMTRDTSTSTVYMELSSLRSEDTAVYYC AR 251 JH4 FR4 WGQGTLVTVSS 252 VH3-30 FR1QVQLVESGGGVVQPGRSLRLSCAASGFTFS 253 VH3-30 FR2 WVRQAPGKGLEWVA 254VH3-30 FR3 RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR 255 JH3 FR4 WGQGTMVTVSS 256VH3 CONSENSUS EVQLVESGGGLVQPGGSLRLSCAASGFTFS FR1 257 VH3 CONSENSUSWVRQAPGKGLEWVS FR2 258 VH3 CONSENSUS RFTISRDNSKNTLYLQMNSLRAEDTAVYYC FR3AR 259 JH3 FR4 WGQGTMVTVSS 260 VH2-70/JH6 FR1EVTLRESGPALVKPTQTLTLTCTFSGFSLS 261 VH2-70/JH6 FR2 WIRQPPGKALEWLA 262VH2-70/JH6 FR3 RLTISKDTSKNQVVLTMTNMDPVDTATYYC AR 263 VH2-70/JH6 FR4WGQGTTVTVSS 264 VH2-26/JH6 FR1 EVTLKESGPVLVKPTETLTLTCTVSGFSLS 265VH2-26/JH6 FR2 WIRQPPGKALEWLA 266 VH2-26/JH6 FR3RLTISKDTSKSQVVLTMTNMDPVDTATYYC AR 267 VH2-26/JH6 FR4 WGQGTTVTVSS 268VH3-72/JH6 FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 269 VH3-72/JH6 FR2WVRQAPGKGLEWVG 270 VH3-72/JH6 FR3 RFTISRDDSKNSLYLQMNSLKTEDTAVYYC AR 271VH3-72/JH6 FR4 WGQGTTVTVSS 272 VH3-21/JH6 FR1EVQLVESGGGLVKPGGSLRLSCAASGFTFS 273 VH3-21/JH6 FR2 WVRQAPGKGLEWVS 274VH3-21/JH6 FR3 RFTISRDNAKNSLYLQMNSLRAEDTAVYYC AR 275 VH3-21/JH6 FR4WGQGTTVTVSS 276 VH1-69/JH6 FR1 EVQLVQSGAEVKKPGSSVKVSCKASGGTFS 277VH1-69/JH6 FR2 WVRQAPGQGLEWMG 278 VH1-69/JH6 FR3RVTITADKSTSTAYMELSSLRSEDTAVYYC AR 279 VH1-69/JH6 FR4 WGQGTTVTVSS 280VH1-18/JH6 FR1 EVQLVQSGAEVKKPGASVKVSCKASGYTFT 281 VH1-18/JH6 FR2WVRQAPGQGLEWMG 282 VH1-18/JH6 FR3 RVTMTTDTSTSTAYMELRSLRSDDTAVYYC AR 283VH1-18/JH6 FR4 WGQGTTVTVSS 284 IGHV4-59 FR1EVQLQESGPGLVKPSETLSLTCTVSGGSIS 285 IGHV4-59 FR2 WIRQPPGKGLEWIG 286IGHV4-59 FR3 RVTISVDTSKNQFSLKLSSVTAADTAVYYC AR 287 IGHV4-59/JH FR4WGQGTLVTVSS 288 IGHV3-66 FR1 EVQLVESGGGLVQPGGSLRLSCAVSGGSIS 289IGHV3-66 FR2 WIRQAPGKGLEWIG 290 IGHV3-66 FR3RVTISVDTSKNSFYLQMNSLRAEDTAVYYC AR 291 IGHV3-66/JH FR4 WGQGTLVTVSS 292IGHV4-59 FR1 EVQLQESGPGLVKPGETLSLTCTVSGGSIS 293 IGHV4-59 FR2WIRQAPGKGLEWIG 294 IGHV4-59 FR3 RVTISVDTSKNQFYLKLSSVRAEDTAVYYC AR 295IGHV4-59/JH FR4 WGQGTLVTVSS 296 IGHV5-51 FR1EVQLVQSGTEVKKPGESLKISCKVSGGSIS 297 IGHV5-51 FR2 WIRQMPGKGLEWIG 298IGHV5-51 FR3 QVTISVDTSFNTFFLQWSSLKASDTAMYYC AR 299 IGHV5-51/JH FR4WGQGTMVTVSS 300 IGHV2-70 FR1 EVTLRESGPALVKPTQTLTLTCTVSGGSIS 301IGHV2-70 FR2 WIRQPPGKGLEWIG 302 IGHV2-70 FR3RVTISVDTSKNQFVLTMTNMDPVDTATYYC AR 303 IGHV2-70/JH FR4 WGQGTTVTVSS 304IGHV3-15 FR1 EVQLLESGGGLVKSGGSLRLSCAASGFTFR 305 IGHV3-15 FR2WVRQAPGKGLEWVA 306 IGHV3-15 FR3 RFTISRDNSKNTLYLQLNSLRAEDTAVYYC AK 307IGHV3-15/JH FR4 WGQGTMVTVSS 308 IGHV3-43 FR1EVQLVESGGGVVQPGGSLRLSCAASGFTFG 309 IGHV3-43 FR2 WVRQAPGKGLEWVA 310IGHV3-43 FR3 RFTISRDNSKNTLYLQLNSLRAEDTAVYYC AK 311 IGHV3-43/JH FR4WGQGTMVTVSS 312 VH1-18/JH6 FR4 WGQGTTVTVSS 313 VH7-4.1/JH6 FR1QVQLVQSGSELKKPGASVKVSCKASGYTFT 314 VH7-4.1/JH6 FR2 WVRQAPGQGLEWMG 315VH7-4.1/JH6 FR3 RFVFSLDTSVSTAYLQISSLKAEDTAVYYC AR 316 VH7-4.1/JH6 FR4WGQGTTVTVSS

TABLE 4  Light Chain Acceptor Sequences SEQ Sequence ID Protein NO:region 123456789012345678901234567890 13 FR1 DIQMTQSPSSLSASVGDRVTITC 14FR2 WYQQTPGKAPKLLIY 15 FR3 GVPSRFSGSGSGTDYTFTISSLQPEDIATY YC 16 FR4FGQGTKLQIT 25 O2 FR1 DIQMTQSPSSLSASVGDRVTITC 317 O2 FR2 WYQQKPGKAPKLLIY318 O2 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATY YC 319 JK2 FR4 FGQGTKLEIK 320L2 FR1 EIVMTQSPATLSVSPGERATLSC 321 L2 FR2 WYQQKPGQAPRLLIY 322 L2 FR3GIPARFSGSGSGTEFTLTISSLQSEDFAVY YC 323 JK2 FR4 FGQGTKLEIK 324 B3/JK4 FR1DIVMTQSPDSLAVSLGERATINC 325 B3/JK4 FR2 WYQQKPGQPPKLLIY 326 B3/JK4 FR3GVPDRFSGSGSGTDFTLTISSLQAEDVAVY YC 327 B3/JK4 FR4 FGGGTKVEIKR 328L2/JK4 FR1 EIVMTQSPATLSVSPGERATLSC 329 L2/JK4 FR2 WYQQKPGQAPRLLIY 330L2/JK4 FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVY YC 331 L2/JK4 FR4 FGGGTKVEIKR332 L15/JK4 FR1 DIQMTQSPSSLSASVGDRVTITC 333 L15/JK4 FR2 WYQQKPEKAPKSLIY334 L15/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATY YC 335 L15/JK4 FR4FGGGTKVEIKR 336 L5/JK4 FR1 DIQMTQSPSSVSASVGDRVTITC 337 L5/JK4 FR2WYQQKPGKAPKLLIY 338 L5/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATY YC 339L5/JK4 FR4 FGGGTKVEIKR 340 IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 341IGLV3-1 FR2 WYQQKPGQSPVLVIY 342 IGLV3-1 FR3GIPERFSGSNSGDTATLTISGTQPMDEADY YC 343 IGLV3-1/JL FR4 FGYGTKVTVL 344IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 345 IGLV3-1 FR2 WYQQKPGQSPVLVIY 346IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQPMDEADY YC 347 IGLV3-1/JL FR4GGGTKLTVLG 348 IGLV3-1 FR1 YELTQPPSVSVSPGQTASITC 349 IGLV3-1 FR2WYQQKPGQSPVLVIY 350 IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQPMDEADY YC 351IGLV3-1/JL FR4 GGGTKLTVLG 352 IGLV3-1 FR1 LYVLTQPPSVSVSPGQTASITC 353IGLV3-1 FR2 WYQQKPGQSPVLVIY 354 IGLV3-1 FR3GIPERFSGSNSGDTATLTISGTQTMDEADY LC 355 IGLV3-1/JL FR4 FGGGTKVTVLG 356IGKV6D-21 FR1 EYVLTQSPDFQSVTPKEKVTITC 357 IGKV6D-21 FR2 WYQQKPDQSPKLVIY358 IGKV6D-21 FR3 GVPSRFSGSNSGDDATLTINSLEAEDAATY YC 359 IGKV6D-21/JK FR4FGQGTKVEIKR 360 IGKV3D-15 FR1 EYVLTQSPATLSVSPGERATLSC 361 IGKV3D-15 FR2WYQQKPGQSPRLVIY 362 IGKV3D-15 FR3 DIPARFSGSNSGDEATLTISSLQSEDFAVY YC 363IGKV3D-15/JK FR4 FGQGTRLEIKR 364 IGKV4-1 FR1 DYVLTQSPDSLAVSLGERATINC 365IGKV4-1 FR2 WYQQKPGQSPKLVIY 366 IGKV4-1 FR3GIPDRFSGSNSGDDATLTISSLQAEDVAVY YC 367 IGKV4-1/JK FR4 FGGGTKVEIKR 368IGLV3-1 FR1 LPVLTQPPSVSVSPGQTASITC 369 IGLV3-1 FR2 WYQQKPGQSPVLVIY 370IGLV3-1 FR3 GIPERFSGSNSGNTATLTISGTQTMDEADY LC 371 IGLV3-1JL FR4/FGGGTKVTVL 372 IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 373 IGLV3-1 FR2WYQQKPGQSPVLVIY 374 IGLV3-1 FR3 GIPERFSGSNSGNTATLTISGTQTMDEADY LC 375IGLV3-1/JL FR4 FGGGTKLTVL 376 1-33/018/JK2 FR1 DIQMTQSPSSLSASVGDRVTITC377 1-33/018/JK2 FR2 WYQQKPGKAPKLLIY 378 1-33/018/JK2 FR3GVPSRFSGSGSGTDFTFTISSLQPEDIATY YC 379 1-33/018/JK2 FR4 FGQGTKLEIKR 3801-33/018/JK4 FR4 FGGGTKVEIKR

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol., 22(3):183-200 (2002); Marchalonis et al., Adv. Exp. Med. Biol., 484: 13-30(2001)). One of the advantages provided by various embodiments of thepresent invention stems from the recognition that germline antibodygenes are more likely than mature antibody genes to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as from a foreign sourcewhen used therapeutically in that species.

As used herein, the term “key” residues refer to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

The term “humanized antibody” refers to antibodies that comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like”, i.e., more similar tohuman germline variable sequences. One type of humanized antibody is aCDR-grafted antibody, in which human CDR sequences are introduced intonon-human VH and VL sequences to replace the corresponding nonhuman CDRsequences. Also “humanized antibody” is an antibody or a variant,derivative, analog or fragment thereof which immunospecifically binds toan antigen of interest and which comprises a framework (FR) regionhaving substantially the amino acid sequence of a human antibody and acomplementary determining region (CDR) having substantially the aminoacid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 80%, at least 85%, at least 90%, at least 95%, atleast 98% or at least 99% identical to the amino acid sequence of anon-human antibody CDR. A humanized antibody comprises substantially allof at least one, and typically two, variable domains (Fab, Fab′,F(ab′)₂, FabC, Fv) in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence. In an embodiment, a humanizedantibody also comprises at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. In someembodiments, a humanized antibody contains both the light chain as wellas at least the variable domain of a heavy chain. The antibody also mayinclude the CH₁, hinge, CH₂, CH₃, and CH4 regions of the heavy chain. Insome embodiments, a humanized antibody only contains a humanized lightchain. In some embodiments, a humanized antibody only contains ahumanized heavy chain. In specific embodiments, a humanized antibodyonly contains a humanized variable domain of a light chain and/orhumanized heavy chain.

A humanized antibody may be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype including withoutlimitation IgG1, IgG2, IgG3, and IgG4. The humanized antibody maycomprise sequences from more than one class or isotype, and particularconstant domains may be selected to optimize desired effector functionsusing techniques well known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In an exemplary embodiment,such mutations, however, will not be extensive. Usually, at least 80%,preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (see, e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family ofimmunoglobulins, each position in the consensus sequence is occupied bythe amino acid occurring most frequently at that position in the family.If two amino acids occur equally frequently, either can be included inthe consensus sequence.

With respect to constructing DVD-Ig or other binding protein molecules,a “linker” is used to denote a single amino acid or a polypeptide(“linker polypeptide”) comprising two or more amino acid residues joinedby peptide bonds and used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see, e.g., Holligeret al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak, R. J.,Structure, 2: 1121-1123 (1994)). Exemplary linkers include, but are notlimited to, GGGGSG (SEQ ID NO:26), GGSGG (SEQ ID NO:27), GGGGSGGGGS (SEQID NO:28), GGSGGGGSG (SEQ ID NO:223), GGSGGGGSGS (SEQ ID NO:29),GGSGGGGSGGGGS (SEQ ID NO:30), GGGGSGGGGSGGGG (SEQ ID NO:31),GGGGSGGGGSGGGGS (SEQ ID NO:32), ASTKGP (SEQ ID NO:33), ASTKGPSVFPLAP(SEQ ID NO:34), TVAAP (SEQ ID NO:35), RTVAAP (SEQ ID NO:224),TVAAPSVFIFPP (SEQ ID NO:36), RTVAAPSVFIFPP (SEQ ID NO:225),AKTTPKLEEGEFSEAR (SEQ ID NO:37), AKTTPKLEEGEFSEARV (SEQ ID NO:38),AKTTPKLGG (SEQ ID NO:39), SAKTTPKLGG (SEQ ID NO:40), SAKTTP (SEQ IDNO:41), RADAAP (SEQ ID NO:42), RADAAPTVS (SEQ ID NO:43), RADAAAAGGPGS(SEQ ID NO:44), RADAAAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:45),SAKTTPKLEEGEFSEARV (SEQ ID NO:46), ADAAP (SEQ ID NO:47), ADAAPTVSIFPP(SEQ ID NO:48), QPKAAP (SEQ ID NO:49), QPKAAPSVTLFPP (SEQ ID NO:50),AKTTPP (SEQ ID NO:51), AKTTPPSVTPLAP (SEQ ID NO:52), AKTTAP (SEQ IDNO:53), AKTTAPSVYPLAP (SEQ ID NO:54), GENKVEYAPALMALS (SEQ ID NO:55),GPAKELTPLKEAKVS (SEQ ID NO:56), and GHEAAAVMQVQYPAS (SEQ ID NO:57).

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter, J. Mol. Biol., 224:487-499 (1992), whichis incorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

As used herein, the term “neutralizing” refers to neutralization of thebiological activity of an antigen (e.g., the cytokine IL-1β when abinding protein specifically binds the antigen. Preferably, aneutralizing binding protein described herein binds to h IL-1β resultingin the inhibition of a biological activity of hIL-1β. Preferably, theneutralizing binding protein binds h IL-1β and reduces a biologicallyactivity of hIL-1β by at least about 20%, 40%, 60%, 80%, 85%, or more.Inhibition of a biological activity of h IL-1β by a neutralizing bindingprotein can be assessed by measuring one or more indicators of h IL-1βbiological activity well known in the art. For example inhibition ofhuman IL-6 secretion by IL-1β induction in HS27 cells.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-h IL-1β antibody that binds to an IL-1β antigen and/or theneutralizing potency of an antibody, for example, an anti-IL-1β antibodywhose binding to h IL-1β inhibits the biological activity of h IL-1β,for example, inhibition of human IL-6 secretion by IL-1β induction inHS27 cells.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. In certain embodiments, an antibody is said to specificallybind an antigen when it preferentially recognizes its target antigen ina complex mixture of proteins and/or macromolecules. Antibodies are saidto “bind to the same epitope” if the antibodies cross-compete (oneprevents the binding or modulating effect of the other). In addition,structural definitions of epitopes (overlapping, similar, identical) areinformative, but functional definitions are often more relevant as theyencompass structural (binding) and functional (modulation, competition)parameters.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson et al., Ann. Biol. Clin., 51: 19-26(1993); Jönsson et al., BioTechniques, 11: 620-627 (1991); Jöhnsson etal., J. Mol. Recognit., 8: 125-131 (1995); and Johnsson et al., Anal.Biochem., 198: 268-277 (1991).

The term “K_(on)” (also “Kon”, “kon”), as used herein, is intended torefer to the on rate constant for association of a binding protein(e.g., an antibody) to an antigen to form an association complex, e.g.,antibody/antigen complex, as is known in the art. The “K_(on)” also isknown by the terms “association rate constant”, or “ka”, as usedinterchangeably herein. This value indicates the binding rate of anantibody to its target antigen or the rate of complex formation betweenan antibody and antigen as is shown by the equation below:

Antibody(“Ab”)+Antigen(“Ag”)→Ab-Ag.

The term “K_(off)” (also “Koff”, “koff”), as used herein, is intended torefer to the off rate constant for dissociation, or “dissociation rateconstant”, of a binding protein (e.g., an antibody) from an associationcomplex (e.g., an antibody/antigen complex) as is known in the art. Thisvalue indicates the dissociation rate of an antibody from its targetantigen or separation of Ab-Ag complex over time into free antibody andantigen as shown by the equation below:

Ab+Ag←Ab-Ag.

The term “K_(D)” (also “K_(d)”), as used herein, is intended to refer tothe “equilibrium dissociation constant”, and refers to the valueobtained in a titration measurement at equilibrium, or by dividing thedissociation rate constant (Koff) by the association rate constant(Kon). The association rate constant (Kon), the dissociation rateconstant (Koff), and the equilibrium dissociation constant (K are usedto represent the binding affinity of an antibody to an antigen. Methodsfor determining association and dissociation rate constants are wellknown in the art. Using fluorescence-based techniques offers highsensitivity and the ability to examine samples in physiological buffersat equilibrium. Other experimental approaches and instruments such as aBIAcore® (biomolecular interaction analysis) assay can be used (e.g.,instrument available from BIAcore International AB, a GE Healthcarecompany, Uppsala, Sweden). Additionally, a KinExA® (Kinetic ExclusionAssay) assay, available from Sapidyne Instruments (Boise, Id.) can alsobe used.

The terms “label” and “detectable label” mean a moiety attached to aspecific binding partner, such as an antibody or an analyte, e.g., torender the reaction between members of a specific binding pair, such asan antibody and an analyte, detectable. The specific binding partner,e.g., antibody or analyte, so labeled is referred to as “detectablylabeled”. Thus, the term “labeled binding protein” as used herein,refers to a protein with a label incorporated that provides for theidentification of the binding protein. In an embodiment, the label is adetectable marker that can produce a signal that is detectable by visualor instrumental means, e.g., incorporation of a radiolabeled amino acidor attachment to a polypeptide of biotinyl moieties that can be detectedby marked avidin or streptavidin (e.g., streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Examples of labels for polypeptides include,but are not limited to, the following: radioisotopes or radionuclides(e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or¹⁵³Sm), chromogens, fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,luciferase, alkaline phosphatase), chemiluminescent markers, biotinylgroups, predetermined polypeptide epitopes recognized by a secondaryreporter (e.g., leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags), and magneticagents (e.g., gadolinium chelates). Representative examples of labelscommonly employed for immunoassays include moieties that produce light,e.g., acridinium compounds, and moieties that produce fluorescence,e.g., fluorescein. Other labels are described herein. In this regard,the moiety itself may not be detectably labeled but may becomedetectable upon reaction with yet another moiety. Use of the term“detectably labeled” is intended to encompass the latter type ofdetectable labeling.

The term “IL-1β binding protein conjugate” refers to an IL-1β bindingprotein described herein chemically linked to a second chemical moiety,such as a therapeutic or cytotoxic agent. The term “agent” is usedherein to denote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Whenemployed in the context of an immunoassay, an IL-1β binding proteinconjugate may be a detectably labeled antibody, which is used as thedetection antibody.

The terms “crystal” and “crystallized” as used herein, refer to abinding protein (e.g., an antibody), or antigen binding portion thereof,that exists in the form of a crystal. Crystals are one form of the solidstate of matter that is distinct from other forms such as the amorphoussolid state or the liquid crystalline state. Crystals are composed ofregular, repeating, three-dimensional arrays of atoms, ions, molecules(e.g., proteins such as antibodies), or molecular assemblies (e.g.,antigen/antibody complexes). These three-dimensional arrays are arrangedaccording to specific mathematical relationships that arewell-understood in the field. The fundamental unit, or building block,that is repeated in a crystal is called the asymmetric unit. Repetitionof the asymmetric unit in an arrangement that conforms to a given,well-defined crystallographic symmetry provides the “unit cell” of thecrystal. Repetition of the unit cell by regular translations in allthree dimensions provides the crystal. See Giegé et al., Chapter 1, InCrystallization of Nucleic Acids and Proteins, a Practical Approach, 2nded., (Ducruix and Giegé, eds.) (Oxford University Press, New York, 1999)pp. 1-16.

The term “polynucleotide” means a polymeric form of two or morenucleotides, either ribonucleotides or deoxynucleotides or a modifiedform of either type of nucleotide. The term includes single and doublestranded forms of DNA.

The term “isolated polynucleotide” shall mean a polynucleotide (e.g., ofgenomic, cDNA, or synthetic origin, or some combination thereof) that,by virtue of its origin, the “isolated polynucleotide” is not associatedwith all or a portion of a polynucleotide with which the “isolatedpolynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences that are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), is intended torefer to a cell into which exogenous DNA has been introduced. In anembodiment, the host cell comprises two or more (e.g., multiple) nucleicacids encoding antibodies, such as the host cells described in U.S. Pat.No. 7,262,028, for example. Such terms are intended to refer not only tothe particular subject cell, but also to the progeny of such a cell.Because certain modifications may occur in succeeding generations due toeither mutation or environmental influences, such progeny may not, infact, be identical to the parent cell, but are still included within thescope of the term “host cell” as used herein. In an embodiment, hostcells include prokaryotic and eukaryotic cells selected from any of theKingdoms of life. In another embodiment, eukaryotic cells includeprotist, fungal, plant and animal cells. In another embodiment, hostcells include but are not limited to the prokaryotic cell lineEscherichia coli; mammalian cell lines CHO, HEK 293, COS, NS0, SP2 andPER.C6; the insect cell line Sf9; and the fungal cell Saccharomycescerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

“Transgenic organism”, as known in the art, refers to an organism havingcells that contain a transgene, wherein the transgene introduced intothe organism (or an ancestor of the organism) expresses a polypeptidenot naturally expressed in the organism. A “transgene” is a DNAconstruct, which is stably and operably integrated into the genome of acell from which a transgenic organism develops, directing the expressionof an encoded gene product in one or more cell types or tissues of thetransgenic organism.

The terms “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of h IL-1β).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of hIL-1β). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies are described, e.g., in PCT Publication No. WO 01/83525.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, causes an increase in themagnitude of a certain activity or function of the molecule compared tothe magnitude of the activity or function observed in the absence of theagonist. Particular agonists of interest may include, but are notlimited to, IL-1β polypeptides, nucleic acids, carbohydrates, or anyother molecule that binds to hIL-1β.

The terms “antagonist” and “inhibitor”, as used herein, refer to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Particular antagonists of interestinclude those that block or modulate the biological or immunologicalactivity of human IL-1β. Antagonists and inhibitors of human IL-1β mayinclude, but are not limited to, proteins, nucleic acids, carbohydrates,or any other molecules, which bind to human IL-1β.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof; prevent theadvancement of a disorder; cause regression of a disorder; prevent therecurrence, development, onset, or progression of one or more symptomsassociated with a disorder; detect a disorder; or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

“Patient” and “subject” may be used interchangeably herein to refer toan animal, such as a mammal, including a primate (for example, a human,a monkey, and a chimpanzee), a non-primate (for example, a cow, a pig, acamel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guineapig, a cat, a dog, a rat, a mouse, a whale), a bird (e.g., a duck or agoose), and a shark. Preferably, a patient or subject is a human, suchas a human being treated or assessed for a disease, disorder orcondition, a human at risk for a disease, disorder or condition, a humanhaving a disease, disorder or condition, and/or human being treated fora disease, disorder or condition.

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans,non-human primates, mice, rats, monkeys, dogs, rabbits and otheranimals. Such substances include, but are not limited to, blood (e.g.,whole blood), plasma, serum, urine, amniotic fluid, synovial fluid,endothelial cells, leukocytes, monocytes, other cells, organs, tissues,bone marrow, lymph nodes and spleen.

“Component”, “components,” and “at least one component,” refer generallyto a capture antibody, a detection or conjugate antibody, a control, acalibrator, a series of calibrators, a sensitivity panel, a container, abuffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, adetection reagent, a pretreatment reagent/solution, a substrate (e.g.,as a solution), a stop solution, and the like that can be included in akit for assay of a test sample, such as a patient urine, serum or plasmasample, in accordance with the methods described herein and othermethods known in the art. Thus, in the context of the presentdisclosure, “at least one component,” “component,” and “components” caninclude a polypeptide or other analyte as above, such as a compositioncomprising an analyte such as polypeptide, which is optionallyimmobilized on a solid support, such as by binding to an anti-analyte(e.g., anti-polypeptide) antibody. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Control” refers to a composition known to not analyte (“negativecontrol”) or to contain analyte (“positive control”). A positive controlcan comprise a known concentration of analyte. “Control,” “positivecontrol,” and “calibrator” may be used interchangeably herein to referto a composition comprising a known concentration of analyte. A“positive control” can be used to establish assay performancecharacteristics and is a useful indicator of the integrity of reagents(e.g., analytes).

“Predetermined cutoff” and “predetermined level” refer generally to anassay cutoff value that is used to assessdiagnostic/prognostic/therapeutic efficacy results by comparing theassay results against the predetermined cutoff/level, where thepredetermined cutoff/level already has been linked or associated withvarious clinical parameters (e.g., severity of disease,progression/nonprogression/improvement, etc.). While the presentdisclosure may provide exemplary predetermined levels, it is well-knownthat cutoff values may vary depending on the nature of the immunoassay(e.g., antibodies employed, etc.). It further is well within theordinary skill of one in the art to adapt the disclosure herein forother immunoassays to obtain immunoassay-specific cutoff values forthose other immunoassays based on this disclosure. Whereas the precisevalue of the predetermined cutoff/level may vary between assays,correlations as described herein (if any) should be generallyapplicable.

“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilizationreagent, as used in a diagnostic assay as described herein is one thatlyses any cells and/or solubilizes any analyte that is/are present in atest sample. Pretreatment is not necessary for all samples, as describedfurther herein. Among other things, solubilizing the analyte (e.g.,polypeptide of interest) may entail release of the analyte from anyendogenous binding proteins present in the sample. A pretreatmentreagent may be homogeneous (not requiring a separation step) orheterogeneous (requiring a separation step). With use of a heterogeneouspretreatment reagent there is removal of any precipitated analytebinding proteins from the test sample prior to proceeding to the nextstep of the assay.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction so as tocomprise a “sensitivity panel.”

“Risk” refers to the possibility or probability of a particular eventoccurring either presently or at some point in the future. “Riskstratification” refers to an array of known clinical risk factors thatallows physicians to classify patients into a low, moderate, high orhighest risk of developing a particular disease, disorder or condition.

“Specific” and “specificity” in the context of an interaction betweenmembers of a specific binding pair (e.g., an antigen (or fragmentthereof) and an antibody (or antigenically reactive fragment thereof))refer to the selective reactivity of the interaction. The phrase“specifically binds to” and analogous phrases refer to the ability ofantibodies (or antigenically reactive fragments thereof) to bindspecifically to analyte (or a fragment thereof) and not bindspecifically to other entities.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs ofcommon immunoassays, other specific binding pairs can include biotin andavidin (or streptavidin), carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors and enzymes, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding members, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments, and antibodies, including monoclonal and polyclonalantibodies as well as complexes, fragments, and variants (includingfragments of variants) thereof, whether isolated or recombinantlyproduced.

“Variant” as used herein means a polypeptide that differs from a givenpolypeptide (e.g., IL-1β, BNP, NGAL, or HIV polypeptide, oranti-polypeptide antibody) in amino acid sequence by the addition (e.g.,insertion), deletion, or conservative substitution of amino acids, butthat retains the biological activity of the given polypeptide (e.g., avariant IL-1β can compete with anti-IL-1β antibody for binding toIL-1β). A conservative substitution of an amino acid, i.e., replacing anamino acid with a different amino acid of similar properties (e.g.,hydrophilicity and degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (see, e.g., Kyte et al., J.Mol. Biol., 157: 105-132 (1982)). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids also can be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.4,554,101). Substitution of amino acids having similar hydrophilicityvalues can result in peptides retaining biological activity, for exampleimmunogenicity, as is understood in the art. In one aspect,substitutions are performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hydrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.“Variant” also can be used to describe a polypeptide or fragment thereofthat has been differentially processed, such as by proteolysis,phosphorylation, or other post-translational modification, yet retainsits biological activity or antigen reactivity, e.g., the ability to bindto IL-1β. Use of “variant” herein is intended to encompass fragments ofa variant unless otherwise contradicted by context.

I. Antibodies that Bind Human IL-1β

One aspect of the present invention provides isolated murine monoclonalantibodies, or antigen-binding portions thereof, that bind to IL-1β withhigh affinity, a slow off rate and high neutralizing capacity. A secondaspect of the invention provides chimeric antibodies that bind IL-1β. Athird aspect of the invention provides CDR grafted antibodies, orantigen-binding portions thereof, that bind IL-1β. A fourth aspect ofthe invention provides humanized antibodies, or antigen-binding portionsthereof, that bind IL-1β. A fifth aspect of the invention provides dualvariable domain immunoglobulin (DVD-Ig™) molecules that bind IL-1β andone other target. Preferably, the antibodies, or portions thereof, areisolated antibodies. Preferably, the antibodies of the invention areneutralizing human anti-IL-1β antibodies.

A. Method of Making Anti-IL-1β Antibodies

Anti-IL-1β antibodies of the present invention may be made by any of anumber of techniques known in the art.

1. Anti IL-1β Monoclonal Antibodies Using Hybridoma Technology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press,1988); Hammerling et al., eds., “Monoclonal Antibodies and T-CellHybridomas,” In Research Monographs in Immunology, vol. 3 (J. L. Turk,General Editor) (Elsevier, New York, 1981) pp. 563-587 (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific anti-IL-1β antibodiesusing hybridoma technology are routine and well known in the art. In oneembodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention. Briefly, mice can be immunized withan IL-1β antigen. In an exemplary embodiment, the IL-1β antigen isadministered with an adjuvant to stimulate the immune response. Suchadjuvants include complete or incomplete Freund's adjuvant, RIBI(muramyl dipeptides) or ISCOM (immunostimulating complexes). Suchadjuvants may protect the polypeptide from rapid dispersal bysequestering it in a local deposit, or they may contain substances thatstimulate the host to secrete factors that are chemotactic formacrophages and other components of the immune system. Preferably, if apolypeptide is being administered, the immunization schedule willinvolve two or more administrations of the polypeptide, spread out overseveral weeks.

After immunization of an animal with an IL-1β antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-IL-1βantibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-IL-1β antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen IL-1β are detected in the mouse serum, the mouse spleen isharvested and splenocytes isolated. The splenocytes are then fused bywell-known techniques to any suitable myeloma cells, for example cellsfrom cell line SP20 available from the American Type Culture Collection(ATCC, Manassas, Va.). Hybridomas are selected and cloned by limiteddilution. The hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding IL-1β. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow et al., supra. Inan exemplary embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using IL-1β, or a portionthereof, or a cell expressing IL-1β. In an exemplary embodiment, theinitial screening is performed using an enzyme-linked immunosorbentassay (ELISA) or a radioimmunoassay (RIA), preferably an ELISA. Anexample of ELISA screening is provided in PCT Publication No. WO00/37504, incorporated herein by reference.

Anti-IL-1β antibody-producing hybridomas are selected, cloned, andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning, and expanding hybridomas are well known to those ofordinary skill in the art.

In an exemplary embodiment, the hybridomas are mouse hybridomas, asdescribed above. In another preferred embodiment, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an anti-IL-1β antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region, and the CH1 domain ofthe heavy chain.

2. Anti-IL-1D Monoclonal Antibodies Using SLAM

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook etal., Proc. Natl. Acad. Sci. USA, 93: 7843-7848 (1996). In this method,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals described in Section 1, arescreened using an antigen-specific hemolytic plaque assay, wherein theantigen IL-1β, a subunit of IL-1β, or a fragment thereof, is coupled tosheep red blood cells using a linker, such as biotin, and used toidentify single cells that secrete antibodies with specificity forIL-1β. Following identification of antibody-secreting cells of interest,heavy and light chain variable region (VH and VL) cDNAs are rescued fromthe cells by reverse transcriptase-PCR, and these variable regions canthen be expressed, in the context of appropriate immunoglobulin constantregions (e.g., human constant regions), in mammalian host cells, such asCOS or CHO cells. The host cells transfected with the amplifiedimmunoglobulin sequences, derived from in vivo selected lymphocytes, canthen undergo further analysis and selection in vitro, for example, bypanning the transfected cells to isolate cells expressing antibodies toIL-1β. The amplified immunoglobulin sequences further can be manipulatedin vitro, such as by in vitro affinity maturation methods such as thosedescribed in PCT Publication No. WO 97/29131 and PCT Publication No. WO00/56772.

3. Anti-IL-1D Monoclonal Antibodies Using Transgenic Animals

In another embodiment of the invention, antibodies are produced byimmunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with an IL-1β antigen. In an exemplary embodiment,the non-human animal is a XENOMOUSE transgenic mouse, an engineeredmouse strain that comprises large fragments of the human immunoglobulinloci and is deficient in mouse antibody production. See, e.g., Green etal., Nature Genetics, 7: 13-21 (1994) and U.S. Pat. Nos. 5,916,771;5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598 and6,130,364. See also PCT Publication Nos. WO 91/10741, published Jul. 25,1991; WO 94/02602, published Feb. 3, 1994; WO 96/34096 and WO 96/33735,both published Oct. 31, 1996; WO 98/16654, published Apr. 23, 1998; WO98/24893, published Jun. 11, 1998; WO 98/50433, published Nov. 12, 1998;WO 99/45031, published Sep. 10, 1999; WO 99/53049, published Oct. 21,1999; WO 00/09560, published Feb. 24, 2000; and WO 00/037504, publishedJun. 29, 2000. The XENOMOUSE® transgenic mouse produces an adult-likehuman repertoire of fully human antibodies, and generatesantigen-specific human Mabs. The XENOMOUSE® transgenic mouse containsapproximately 80% of the human antibody repertoire through introductionof megabase sized, germline configuration YAC fragments of the humanheavy chain loci and x light chain loci. See, Mendez et al., NatureGenetics, 15:146-156 (1997); and Green and Jakobovits, J. Exp. Med.,188: 483-495 (1998), the disclosures of which are hereby incorporated byreference.

4. Anti-IL-1D Monoclonal Antibodies Using Recombinant Antibody Libraries

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, Ladner et al., U.S. Pat.No. 5,223,409; Kang et al., PCT Publication No. WO 92/18619; Dower etal., PCT Publication No. WO 91/17271; Winter et al., PCT Publication No.WO 92/20791; Markland et al., PCT Publication No. WO 92/15679; Breitlinget al., PCT Publication No. WO 93/01288; McCafferty et al., PCTPublication No. WO 92/01047; Garrard et al., PCT Publication No. WO92/09690; Fuchs et al., Bio/Technology, 9: 1369-1372 (1991); Hay et al.,Hum. Antibod. Hybridomas, 3: 81-85 (1992); Huse et al., Science, 246:1275-1281 (1989); McCafferty et al., Nature, 348: 552-554 (1990);Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkins et al., J. Mol.Biol., 226: 889-896 (1992); Clackson et al., Nature, 352: 624-628(1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89: 3576-3580 (1992);Garrard et al., Bio/Technology, 9: 1373-1377 (1991); Hoogenboom et al.,Nucl. Acids Res., 19: 4133-4137 (1991); and Barbas et al., Proc. Natl.Acad. Sci. USA, 88: 7978-7982 (1991); US Publication No. 2003/0186374;and PCT Publication No. WO 97/29131, the contents of each of which areincorporated herein by reference.

The recombinant antibody library may be from a subject immunized withIL-1β, or a portion of IL-1β. Alternatively, the recombinant antibodylibrary may be from a naïve subject, i.e., one who has not beenimmunized with IL-1β, such as a human antibody library from a humansubject who has not been immunized with human IL-1β. Antibodies of theinvention are selected by screening the recombinant antibody librarywith the peptide comprising human IL-1β to thereby select thoseantibodies that recognize IL-1β. Methods for conducting such screeningand selection are well known in the art, such as described in thereferences in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for human IL-1β, such asthose that dissociate from human IL-1β with a particular K_(off) rateconstant, the art-known method of surface plasmon resonance can be usedto select antibodies having the desired K_(off) rate constant. To selectantibodies of the invention having a particular neutralizing activityfor human IL-1β, such as those with a particular an IC₅₀, standardmethods known in the art for assessing the inhibition of human IL-1βactivity may be used.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human IL-1β. Preferably, theantibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv, or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmannet al., J. Immunol. Methods, 182: 41-50 (1995); Ames et al., J. Immunol.Methods, 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol.,24: 952-958 (1994); Persic et al., Gene, 187: 9-18 (1997); Burton etal., Adv. Immunol., 57: 191-280 (1994); PCT Publications Nos. WO90/02809; WO 91/10737; WO 92/01047 (PCT/GB91/01134); WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTPublication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); and Sawai et al., Am. J. Reprod. Immunol., 34: 26-34(1995); and Better et al., Science, 240: 1041-1043 (1988), (saidreferences incorporated by reference in their entireties). Examples oftechniques which can be used to produce single-chain Fvs and antibodiesinclude those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;Huston et al., Methods Enzymol., 203: 46-88 (1991); Shu et al., Proc.Natl. Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al., Science,240: 1038-1041 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of dualspecificity antibodies of the invention. One type of alternativeexpression system is one in which the recombinant antibody library isexpressed as RNA-protein fusions, as described in PCT Publication No. WO98/31700 by Szostak and Roberts; and in Roberts and Szostak, Proc. Natl.Acad. Sci. USA, 94: 12297-12302 (1997). In this system, a covalentfusion is created between an mRNA and the peptide or protein that itencodes by in vitro translation of synthetic mRNAs that carry puromycin,a peptidyl acceptor antibiotic, at their 3′ end. Thus, a specific mRNAcan be enriched from a complex mixture of mRNAs (e.g., a combinatoriallibrary) based on the properties of the encoded peptide or protein,e.g., antibody, or portion thereof, such as binding of the antibody, orportion thereof, to the dual specificity antigen. Nucleic acid sequencesencoding antibodies, or portions thereof, recovered from screening ofsuch libraries can be expressed by recombinant means as described above(e.g., in mammalian host cells) and, moreover, can be subjected tofurther affinity maturation by either additional rounds of screening ofmRNA-peptide fusions in which mutations have been introduced into theoriginally selected sequence(s), or by other methods for affinitymaturation in vitro of recombinant antibodies, as described above.

In another approach, the antibodies of the present invention can also begenerated using yeast display methods known in the art. In yeast displaymethods, genetic methods are used to tether antibody domains to theyeast cell wall and display them on the surface of yeast. In particular,such yeast can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Examples of yeast display methods that can be used to make theantibodies of the present invention include those disclosed Wittrup etal. in U.S. Pat. No. 6,699,658, incorporated herein by reference.

B. Production of Recombinant IL-1β Antibodies

Antibodies of the present invention may be produced by any of a numberof techniques known in the art. For example, expression from host cells,wherein expression vector(s) encoding the heavy and light chains is(are) transfected into a host cell by standard techniques. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection, and thelike. Although it is possible to express the antibodies of the inventionin either prokaryotic or eukaryotic host cells, expression of antibodiesin eukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982)), NS0myeloma cells, COS cells and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theantigens of interest by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In an exemplary system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

1. Anti-Human IL-1β Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. Methods for producing chimericantibodies are known in the art and discussed in detail in the Examplessection. See, e.g., Morrison, S. L., Science, 229: 1202-1207 (1985); Oiet al., BioTechniques, 4: 214-221 (1986); Gillies et al., J. Immunol.Methods, 125: 191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and4,816,397, which are incorporated herein by reference in theirentireties. In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Neuberger et al., Nature, 312: 604-608 (1984); Takedaet al., Nature, 314: 452-454 (1985), which are incorporated herein byreference in their entireties) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.

In one embodiment, the chimeric antibodies of the invention are producedby replacing the heavy chain constant region of the murine monoclonalanti-human IL-1β antibodies described in section 1 with a human IgG1constant region.

2. Anti-IL-1β CDR-Grafted Antibodies

CDR-grafted antibodies of the invention comprise heavy and light chainvariable region sequences from a human antibody wherein one or more ofthe CDR regions of V_(H) and/or V_(L) are replaced with CDR sequences ofthe murine antibodies of the invention. A framework sequence from anyhuman antibody may serve as the template for CDR grafting. However,straight chain replacement onto such a framework often leads to someloss of binding affinity to the antigen. The more homologous a humanantibody is to the original murine antibody, the less likely thepossibility that combining the murine CDRs with the human framework willintroduce distortions in the CDRs that could reduce affinity. Therefore,it is preferable that the human variable framework that is chosen toreplace the murine variable framework apart from the CDRs have at leasta 65% sequence identity with the murine antibody variable regionframework. It is more preferable that the human and murine variableregions apart from the CDRs have at least 70% sequence identity. It iseven more preferable that the human and murine variable regions apartfrom the CDRs have at least 75% sequence identity. It is most preferablethat the human and murine variable regions apart from the CDRs have atleast 80% sequence identity. Methods for producing chimeric antibodiesare known in the art. See, for example, European Patent No. EP 0 239400; PCT Publication No. WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089). For veneering or resurfacing of antibodies,see, for example, European Patent Nos. EP 0 592 106 and EP 0 519 596;Padlan, Mol. Immunol., 28(4/5): 489-498 (1991); Studnicka et al.,Protein Eng., 7(6): 805-814 (1994); and Roguska et al., Proc. Natl.Acad. Sci. USA, 91: 969-973 (1994)). Regarding antibody chain shuffling,see, for example, U.S. Pat. No. 5,565,352.

3. Anti-Human IL-1β Humanized Antibodies

Humanized antibodies are antibody molecules derived from non-humanspecies antibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesantibody and framework regions from a human immunoglobulin molecule.Known human Ig sequences are disclosed, e.g., at worldwide web sites:www.ncbi.nlm.nih.gov/entrez-/query.fcgi; www.atcc.org/phage/hdb.html;www.sciquest.com/; www.abcam.com/;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/.about.pedro/research_tools.html;www.mgen.uni-heidelberg.de/SD/IT/IT.html;www.whfreeman.com/immunology/CH-05/kuby05.htm;www.library.thinkquest.org/12429/Immune/Antibody.html;www.hhmi.org/grants/lectures/1996/vlab/;www.path.cam.ac.uk/.about.mrc7/m-ikeimages.html;www.antibodyresource.com/; mcb.harvard.edu/BioLinks/Immuno-logy.html.www.immunologylink.com/; pathbox.wustl.edu/.about.hcenter/index.-html;www.biotech.ufl.eduLabout.hcl/; www.pebio.com/pa/340913/340913.html-;www.nal.usda.gov/awic/pubs/antibody/;www.m.ehime-u.acjp/.about.yasuhito-/Elisa.html;www.biodesign.com/table.asp; www.icnet.uk/axp/facs/davies/lin-ks.html;www.biotech.ufl.eduLabout.fccl/protocol.html;www.isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.n1/.about.jraats/linkslhtml;www.recab.uni-hd.de/immuno.bme.nwu.edu/;www.mrc-cpe.cam.ac.uk/imt-doc/pu-blic/INTRO.html;www.ibt.unam.mx/vir/V_mice.html; imgt.cnusc.fr:8104/;www.biochem.ucl.ac.uk/.about.martin/abs/index.html;antibody.bath.ac.uk/; abgen.cvm.tamu.edu/lab/wwwabgen.html;www.unizh.ch/.about.honegger/AHOsem-inar/Slide01.html;www.cryst.bbk.ac.uk/.about.ubcg07s/;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;www.path.cam.ac.uk/.about.mrc7/h-umanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html;www.cryst.bioc.cam.ac.uk/.abo-ut.fmolina/Web-pages/Pept/spottech.html;www.jerini.de/fr roducts.htm; www.patents.ibm.com/ibm.html.Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework (FR) residues in the human framework regions may besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature, 332: 323-327 (1988), which are incorporated herein byreference in their entireties. Three-dimensional immunoglobulin modelsare commonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. Antibodies can be humanized using a varietyof techniques known in the art, such as but not limited to thosedescribed in Jones et al., Nature, 321:522-525 (1986); Verhoeyen et al.,Science, 239:1534-1536 (1988); Sims et al., J. Immunol., 151: 2296-2308(1993); Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987), Carter etal., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992); Presta et al., J.Immunol., 151: 2623-2632 (1993); Padlan, Mol. Immunol., 28(4/5): 489-498(1991); Studnicka et al., Protein Eng., 7(6): 805-814 (1994); Roguska etal., Proc. Natl. Acad. Sci. USA, 91: 969-973 (1994); PCT PublicationNos. WO 91/09967; WO 90/14443; WO 90/14424; WO 90/14430; WO 99/06834(PCT/US98/16280); WO 97/20032 (PCT/US96/18978); WO 92/11272(PCT/US91/09630); WO 92/03461 (PCT/US91/05939); WO 94/18219(PCT/US94/01234); WO 92/01047 (PCT/GB91/01134); and WO 93/06213(PCT/GB92/01755); European Patent Nos. EP 0 592 106; EP 0 519 596 and EP0 239 400; U.S. Pat. Nos. 5,565,332; 5,723,323; 5,976,862; 5,824,514;5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352;6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539 and4,816,567, each entirely incorporated herein by reference, includedreferences cited therein.

5. Anti IL-1β DVD-Ig™ Binding Proteins

Also provided are dual variable domain immunoglobulin binding proteins(DVD-Igs) that bind one or more epitopes of IL-1β. A DVD-Ig bindingprotein may also bind an epitope of IL-1β and an epitope of a secondtarget antigen other than an IL-1β polypeptide. An exemplary embodimentof such DVD-Ig molecules comprises a heavy chain that comprises thestructural formula VD1-(X1)n-VD2-C—(X2)n, wherein VD1 is a first heavychain variable domain, VD2 is a second heavy chain variable domain, C isa heavy chain constant domain, X1 is a linker with the proviso that itis not CH₁, X2 is an Fc region, and n is 0 or 1, and preferably 1; and alight chain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first light chain variable domain, VD2 is a secondlight chain variable domain, C is a light chain constant domain, X1 is alinker with the proviso that it is not CH1, and X2 does not comprise anFc region; and n is 0 or 1, and preferably 1. Such a DVD-Ig may comprisetwo such heavy chains and two such light chains, wherein each chaincomprises variable domains linked in tandem without an interveningconstant region between variable regions, wherein a heavy chain and alight chain associate to form two tandem antigen binding sites, and apair of heavy and light chains may associate with another pair of heavyand light chains to form a tetrameric binding protein with four antigenbinding sites. In another embodiment, a DVD-Ig molecule may compriseheavy and light chains that each comprise three variable domains, e.g.,VD1, VD2, VD3, linked in tandem without an intervening constant regionbetween variable domains, wherein a pair of heavy and light chains mayassociate to form three antigen binding sites, and wherein a pair ofheavy and light chains may associate with another pair of heavy andlight chains to form a tetrameric binding protein with six antigenbinding sites.

Each variable domain (VD) in a DVD-Ig may be obtained from one or more“parent” monoclonal antibodies that bind one or more desired antigens orepitopes, such as IL-1β and/or IL-1α antigens or epitopes.

A. Generation of Parent Monoclonal Antibodies

The variable domains of the DVD-Ig binding protein can be obtained fromparent antibodies, including monoclonal antibodies (mAb), capable ofbinding antigens of interest. These antibodies may be naturallyoccurring or may be generated by recombinant technology. It isunderstood that if an antibody that binds a desired target antigen orepitope is polyclonal then it is still necessary to obtain the variabledomains of an antigen binding site of a single antibody from thepolyclonal population, i.e., of a single monoclonal member of thepolyclonal population, for use in generating a DVD-Ig. Monoclonalantibodies may be generated by any of variety of methods known in theart, including those described herein (see, sections A.1.-A.4., above).

B. Criteria for Selecting Parent Monoclonal Antibodies

An embodiment of the invention pertains to selecting parent antibodieswith at least one or more properties desired in the DVD-Ig molecule. Inan embodiment, the desired property is selected from one or moreantibody parameters. In another embodiment, the antibody parameters areselected from the group consisting of antigen specificity, affinity toantigen, potency, biological function, epitope recognition, stability,solubility, production efficiency, immunogenicity, pharmacokinetics,bioavailability, tissue cross reactivity, and orthologous antigenbinding.

B1. Affinity to Antigen

The desired affinity of a therapeutic mAb may depend upon the nature ofthe antigen, and the desired therapeutic end-point. In an embodiment,monoclonal antibodies have higher affinities (Kd=0.01-0.50 pM) whenblocking a cytokine-cytokine receptor interaction as such interactionare usually high affinity interactions (e.g., <pM-<nM ranges). In suchinstances, the mAb affinity for its target should be equal to or betterthan the affinity of the cytokine (ligand) for its receptor. On theother hand, mAb with lesser affinity (>nM range) could betherapeutically effective, e.g., in clearing circulating potentiallypathogenic proteins, e.g., monoclonal antibodies that bind to,sequester, and clear circulating species of a target antigen, such asA-β amyloid. In other instances, reducing the affinity of an existinghigh affinity mAb by site-directed mutagenesis or using a mAb with loweraffinity for its target could be used to avoid potential side-effects,e.g., a high affinity mAb may sequester or neutralize all of itsintended target, thereby completely depleting/eliminating thefunction(s) of the targeted protein. In this scenario, a low affinitymAb may sequester/neutralize a fraction of the target that may beresponsible for the disease symptoms (the pathological or over-producedlevels), thus allowing a fraction of the target to continue to performits normal physiological function(s). Therefore, it may be possible toreduce the Kd to adjust dose and/or reduce side-effects. The affinity ofthe parental mAb might play a role in appropriately targeting cellsurface molecules to achieve desired therapeutic out-come. For example,if a target is expressed on cancer cells with high density and on normalcells with low density, a lower affinity mAb will bind a greater numberof targets on tumor cells than normal cells, resulting in tumor cellelimination via ADCC or CDC, and therefore might have therapeuticallydesirable effects. Thus, selecting a mAb with desired affinity may berelevant for both soluble and surface targets.

Signaling through a receptor upon interaction with its ligand may dependupon the affinity of the receptor-ligand interaction. Similarly, it isconceivable that the affinity of a mAb for a surface receptor coulddetermine the nature of intracellular signaling and whether the mAb maydeliver an agonist or an antagonist signal. The affinity-based nature ofmAb-mediated signaling may have an impact of its side-effect profile.Therefore, the desired affinity and desired functions of therapeuticmonoclonal antibodies need to be determined carefully by in vitro and invivo experimentation.

The desired Kd of a binding protein (e.g., an antibody) may bedetermined experimentally depending on the desired therapeutic outcome.In an embodiment, parent antibodies are selected that have an affinity(Kd) for a particular antigen equal to or better than the desiredaffinity of the DVD-Ig for the same antigen. The antigen bindingaffinity and kinetics are assessed by Biacore or another similartechnique. In one embodiment, each parent antibody has a dissociationconstant (Kd) to its antigen selected from the group consisting of: atmost about 10⁻⁷ M; at most about 10⁻⁸ M; at most about 10⁻⁹ M; at mostabout 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about 10⁻¹² M; and at most10⁻¹³M. First parent antibody from which VD1 is obtained and secondparent antibody from which VD2 is obtained may have similar or differentaffinity (K_(D)) for the respective antigen. Each parent antibody has anon rate constant (Kon) to the antigen selected from the group consistingof: at least about 10² M⁻¹ s⁻¹; at least about 10³ M⁻¹ s⁻¹; at leastabout 10⁴ M⁻¹ s⁻¹; at least about 10⁵ M⁻¹ s⁻¹; and at least about 10⁶M⁻¹ s⁻¹, as measured by surface plasmon resonance. The first parentantibody from which, for example, a VD1 is obtained and the secondparent antibody from which a VD2 is obtained may have similar ordifferent on rate constant (Kon) for the respective antigen. In oneembodiment, each parent antibody has an off rate constant (Koff) to theantigen selected from the group consisting of: at most about 10⁻³ s⁻¹;at most about 10⁻⁴ s⁻¹; at most about 10⁻⁵ s⁻¹; and at most about 10⁻⁶s⁻¹, as measured by surface plasmon resonance. The first parent antibodyfrom which VD1 is obtained and the second parent antibody from which VD2is obtained may have similar or different off rate constants (Koff) forthe respective antigen.

B2. Potency

The desired affinity/potency of parental monoclonal antibodies willdepend on the desired therapeutic outcome. For example, forreceptor-ligand (R-L) interactions the affinity (kd) is equal to orbetter than the R-L kd (pM range). For simple clearance of pathologiccirculating proteins, the Kd could be in low nM range, e.g., clearanceof various species of circulating A-β peptide. In addition, the Kd willalso depend on whether the target expresses multiple copies of the sameepitope, e.g., an mAb targeting conformational epitope in Aβ oligomers.

Where VD1 and VD2 bind the same antigen, but distinct epitopes, theDVD-Ig will contain binding sites for the same antigen, thus increasingavidity and thereby the apparent Kd of the DVD-Ig. In an embodiment,parent antibodies with equal or lower Kd than that desired in the DVD-Igare chosen. The affinity considerations of a parental mAb may alsodepend upon whether the DVD-Ig contains four or more identical antigenbinding sites (i.e., a DVD-Ig from a single mAb). In this case, theapparent Kd would be greater than the mAb due to avidity. Such DVD-Igscan be employed for cross-linking surface receptor, increasedneutralization potency, enhanced clearance of pathological proteins,etc.

In another embodiment, parent antibodies with neutralization potency forspecific antigen equal to or better than the desired neutralizationpotential of the DVD-Ig for the same antigen are selected. Theneutralization potency can be assessed by a target-dependent bioassaywhere cells of appropriate type produce a measurable signal (i.e.,proliferation or cytokine production) in response to target stimulation,and target neutralization by the mAb can reduce the signal in adose-dependent manner.

B3. Biological Functions

Monoclonal antibodies can perform potentially several functions. Some ofthese functions are listed in Table 5. These functions can be assessedby both in vitro assays (e.g., cell-based and biochemical assays) and invivo animal models.

TABLE 5 Some Potential Applications for Therapeutic Antibodies Target(Class) Mechanism of Action (target) Soluble Neutralization of activity(e.g., a cytokine, such IL-1β) (cytokines,other) Enhance clearance(e.g., Aβ oligomers) Increase half-life (e.g., GLP 1) Cell SurfaceAgonist (e.g., GLP1 R, EPO R, etc.) (Receptors, other) Antagonist (e.g.,integrins, etc.) Cytotoxic (CD 20, etc.) Protein deposits Enhanceclearance/degradation (e.g., Aβ plaques, amyloid deposits)

MAbs with distinct functions described in the examples herein and inTable 5 can be selected to achieve desired therapeutic outcomes. Two ormore selected parent monoclonal antibodies can then be used in DVD-Igformat to achieve two distinct functions in a single DVD-Ig molecule.For example, a DVD-Ig can be generated by selecting a parent mAb thatneutralizes function of a specific cytokine, such as IL-1β, andselecting a parent mAb that enhances clearance of a pathologicalprotein. Similarly, two parent mAbs may be selected that recognize twodifferent cell surface receptors, one mAb with an agonist function onone receptor and the other mAb with an antagonist function on adifferent receptor. These two selected mAbs, each with a distinctfunction, can be used to construct a single DVD-Ig molecule that willpossess the two distinct functions (agonist and antagonist) of theselected monoclonal antibodies in a single molecule. Similarly, twoantagonistic mAbs to cell surface receptors, each blocking binding ofrespective receptor ligands (e.g., EGF and IGF), may be used in a DVD-Igformat. Conversely, an antagonistic anti-receptor mAb (e.g., anti-EGFR)and a neutralizing anti-soluble mediator (e.g., anti-IGF1/2) mAb can beselected to make a DVD-Ig.

B4. Epitope Recognition:

Different regions of proteins may perform different functions. Forexample, specific regions of a cytokine, such as IL-1β, interact withthe cytokine receptor to bring about receptor activation whereas otherregions of the protein may be required for stabilizing the cytokine. Inthis instance, one may select a mAb that binds specifically to thereceptor interacting region(s) on the cytokine and thereby blockcytokine-receptor interaction. In some cases, for example certainchemokine receptors that bind multiple ligands, a mAb that binds to theepitope (region on chemokine receptor) that interacts with only oneligand can be selected. In other instances, monoclonal antibodies canbind to epitopes on a target that are not directly responsible forphysiological functions of the protein, but binding of a mAb to theseregions could either interfere with physiological functions (sterichindrance) or alter the conformation of the protein such that theprotein cannot function (mAb to receptors with multiple ligand whichalter the receptor conformation such that none of the ligand can bind).Anti-cytokine monoclonal antibodies that do not block binding of thecytokine to its receptor, but block signal transduction have also beenidentified (e.g., 125-2H, an anti-IL-18 mAb).

Examples of epitopes and mAb functions include, but are not limited to,blocking Receptor-Ligand (R-L) interaction (neutralizing mAb that bindsR-interacting site); steric hindrance resulting in diminished or noR-binding. An antibody can bind the target at a site other than areceptor binding site, but still interfere with receptor binding andfunctions of the target by inducing conformational change and eliminatefunction (e.g., XOLAIR® omalizumab, Genetech/Novartis), binding to R butblock signaling (125-2H mAb).

In an embodiment, the parental mAb needs to target the appropriateepitope for maximum efficacy. Such epitope should be conserved in theDVD-Ig. The binding epitope of a mAb can be determined by severalapproaches, including co-crystallography, limited proteolysis ofmAb-antigen complex plus mass spectrometric peptide mapping (Legros etal., Protein Sci., 9: 1002-1010 (2000)), phage displayed peptidelibraries (O'Connor et al., J. Immunol. Methods., 299: 21-35 (2005)), aswell as mutagenesis (Wu C. et al., J. Immunol., 170: 5571-5577 (2003)).

B5. Physicochemical and Pharmaceutical Properties

Therapeutic treatment with antibodies often requires administration ofhigh doses, often several mg/kg (due to a low potency on a mass basis asa consequence of a typically large molecular weight). In order toaccommodate patient compliance and to adequately address chronic diseasetherapies and outpatient treatment, subcutaneous (s.c.) or intramuscular(i.m.) administration of therapeutic mAbs is desirable. For example, themaximum desirable volume for s.c. administration is ˜1.0 mL, andtherefore, concentrations of >100 mg/mL are desirable to limit thenumber of injections per dose. In an embodiment, the therapeuticantibody is administered in one dose. The development of suchformulations is constrained, however, by protein-protein interactions(e.g., aggregation, which potentially increases immunogenicity risks)and by limitations during processing and delivery (e.g., viscosity).Consequently, the large quantities required for clinical efficacy andthe associated development constraints limit full exploitation of thepotential of antibody formulation and s.c. administration in high-doseregimens. It is apparent that the physicochemical and pharmaceuticalproperties of a protein molecule and the protein solution are of utmostimportance, e.g., stability, solubility and viscosity features.

B5.1. Stability

A “stable” antibody formulation is one in which the antibody thereinessentially retains its physical stability and/or chemical stabilityand/or biological activity upon storage. Stability can be measured at aselected temperature for a selected time period. In an embodiment, theantibody in the formulation is stable at room temperature (about 30° C.)or at 40° C. for at least 1 month and/or stable at about 2-8° C. for atleast 1 year, e.g., for at least 2 years. Furthermore, in an embodiment,the formulation is stable following freezing (to, e.g., −70° C.) andthawing of the formulation, hereinafter referred to as a “freeze/thawcycle.” In another example, a “stable” formulation may be one whereinless than about 10% and less than about 5% of the protein is present asan aggregate in the formulation.

A DVD-Ig stable in vitro at various temperatures for an extended timeperiod is desirable. One can achieve this by rapid screening of parentalmAbs stable in vitro at elevated temperature, e.g., at 40° C. for 2-4weeks, and then assess stability. During storage at 2-8° C., the proteinreveals stability for at least 12 months, e.g., at least 24 months.Stability (% of monomeric, intact molecule) can be assessed usingvarious techniques such as cation exchange chromatography, sizeexclusion chromatography, SDS-PAGE, as well as bioactivity testing. Fora more comprehensive list of analytical techniques that may be employedto analyze covalent and conformational modifications, see, Jones, A. J.S., “Analytical methods for the assessment of protein formulations anddelivery systems,” Chapter 2, In Formulation and delivery of peptidesand proteins, 1st ed., (Cleland and Langer, eds.) (American ChemicalSociety, Washington, D.C., 1994) pp. 22-45; and Pearlman and Nguyen,“Analysis of protein drugs,” Chapter 6, In Peptide and protein drugdelivery, 1st ed. [In Advances in Parenteral Sciences, vol. 4] (Lee, V.H., ed.) (Marcel Dekker, Inc., New York, 1991) pp. 247-301.

Heterogeneity and aggregate formation: stability of the antibody may besuch that the formulation may reveal less than about 10%, and, in anembodiment, less than about 5%, in another embodiment, less than about2%, or, in an embodiment, within the range of 0.5% to 1.5% or less inthe GMP antibody material that is present as aggregate. Size exclusionchromatography is a method that is sensitive, reproducible, and veryrobust in the detection of protein aggregates.

In addition to low aggregate levels, the antibody must, in anembodiment, be chemically stable. Chemical stability may be determinedby ion exchange chromatography (e.g., cation or anion exchangechromatography), hydrophobic interaction chromatography, or othermethods such as isoelectric focusing or capillary electrophoresis. Forinstance, chemical stability of the antibody may be such that afterstorage of at least 12 months at 2-8° C. the peak representingunmodified antibody in a cation exchange chromatography may increase notmore than 20%, in an embodiment, not more than 10%, or, in anotherembodiment, not more than 5% as compared to the antibody solution priorto storage testing.

In an embodiment, the parent antibodies display structural integrity;correct disulfide bond formation, and correct folding: Chemicalinstability due to changes in secondary or tertiary structure of anantibody may impact antibody activity. For instance, stability asindicated by activity of the antibody may be such that after storage ofat least 12 months at 2-8° C. the activity of the antibody may decreasenot more than 50%, in an embodiment not more than 30%, or even not morethan 10%, or in an embodiment not more than 5% or 1% as compared to theantibody solution prior to storage testing. Suitable antigen-bindingassays can be employed to determine antibody activity.

B5.2. Solubility

The “solubility” of a mAb correlates with the production of correctlyfolded, monomeric IgG. The solubility of the IgG may therefore beassessed by HPLC. For example, soluble (monomeric) IgG will give rise toa single peak on the HPLC chromatograph, whereas insoluble (e.g.,multimeric and aggregated) will give rise to a plurality of peaks. Aperson skilled in the art will therefore be able to detect an increaseor decrease in solubility of an IgG using routine HPLC techniques. For amore comprehensive list of analytical techniques that may be employed toanalyze solubility (see, Jones, A. G., Dep. Chem. Biochem. Eng., Univ.Coll. London, “Particle formation and separation in suspensioncrystallization processes,” Chapter 4, In Process. Solid-LiquidSuspensions, (P. Ayazi Shamlou, ed.) (Butterworth-Heinemann, Oxford, UK,1993) pp. 93-117; and Pearlman and Nguyen, “Analysis of protein drugs,”Chapter 6, In Peptide and protein drug delivery, 1st ed. [In Advances inParenteral Sciences, vol. 4] (Lee, V. H., ed.) (Marcel Dekker, Inc., NewYork, 1991) pp. 247-301). Solubility of a therapeutic mAb is criticalfor formulating to high concentration often required for adequatedosing. As outlined herein, solubilities of >100 mg/mL may be requiredto accommodate efficient antibody dosing. For instance, antibodysolubility may be not less than about 5 mg/mL in early research phase,in an embodiment not less than about 25 mg/mL in advanced processscience stages, or in an embodiment not less than about 100 mg/mL, or inan embodiment not less than about 150 mg/mL. The intrinsic properties ofa protein molecule are important to the physico-chemical properties ofthe protein solution, e.g., stability, solubility, viscosity. However, aperson skilled in the art will appreciate that a broad variety ofexcipients exist that may be used as additives to beneficially impactthe characteristics of the final protein formulation. These excipientsmay include: (i) liquid solvents, cosolvents (e.g., alcohols such asethanol); (ii) buffering agents (e.g., phosphate, acetate, citrate,amino acid buffers); (iii) sugars or sugar alcohols (e.g., sucrose,trehalose, fructose, raffinose, mannitol, sorbitol, dextrans); (iv)surfactants (e.g., polysorbate 20, 40, 60, 80, poloxamers); (v)isotonicity modifiers (e.g., salts such as NaCl, sugars, sugaralcohols); and (vi) others (e.g., preservatives, chelating agents,antioxidants, chelating substances (e.g., EDTA), biodegradable polymers,carrier molecules (e.g., HSA, PEGs))

Viscosity is a parameter of high importance with regard to antibodymanufacture and antibody processing (e.g.,diafiltration/ultrafiltration), fill-finish processes (pumping aspects,filtration aspects) and delivery aspects (syringeability, sophisticateddevice delivery). Low viscosities enable the liquid solution of theantibody having a higher concentration. This enables the same dose to beadministered in smaller volumes. Small injection volumes provide theadvantage of lower pain during injection, and the solutions do notnecessarily have to be isotonic to reduce pain on injection in thepatient. The viscosity of the antibody solution may be such that atshear rates of 100 (1/s) antibody solution viscosity is below 200 mPa s,in an embodiment below 125 mPa s, in another embodiment below 70 mPa s,and in yet another embodiment below 25 mPa s or even below 10 mPa s.

B5.3. Production Efficiency

The generation of a DVD-Ig that is efficiently expressed in mammaliancells, such as Chinese hamster ovary cells (CHO), will in an embodimentrequire two parental monoclonal antibodies which are themselvesexpressed efficiently in mammalian cells. The production yield from astable mammalian line (i.e., CHO) should be above about 0.5 g/L, in anembodiment above about 1 g/L, and in another embodiment in the range ofabout 2 to about 5 g/L or more (Kipriyanov et al., Mol. Biotechnol.,12:173-201 (1999); Carroll et al., Expert Opin Biol Ther., 4:1821-1829(2004)).

Production of antibodies and Ig fusion proteins in mammalian cells isinfluenced by several factors. Engineering of the expression vector viaincorporation of strong promoters, enhancers and selection markers canmaximize transcription of the gene of interest from an integrated vectorcopy. The identification of vector integration sites that are permissivefor high levels of gene transcription can augment protein expressionfrom a vector (Wurm, F. M., Nature Biotechnol., 22(11): 1393-1398(2004)). Furthermore, levels of production are affected by the ratio ofantibody heavy and light chains and various steps in the process ofprotein assembly and secretion (Jiang et al., Biotechnol. Prog., 22(1):313-318 (2006)).

B6. Immunogenicity

Administration of a therapeutic mAb may result in certain incidence ofan immune response (i.e., the formation of endogenous antibodiesdirected against the therapeutic mAb). Potential elements that mightinduce immunogenicity should be analyzed during selection of theparental monoclonal antibodies, and steps to reduce such risk can betaken to optimize the parental monoclonal antibodies prior to DVD-Igconstruction. Mouse-derived antibodies have been found to be highlyimmunogenic in patients. The generation of chimeric antibodies comprisedof mouse variable and human constant regions presents a logical nextstep to reduce the immunogenicity of therapeutic antibodies (Morrisonand Schlom, “Recombinant Chimeric Monoclonal Antibodies,” Chapter 1, InImportant Advances in Oncology 1990 (J. B. Lippincott Company,Philadelphia, 1990) pp. 3-18). Alternatively, immunogenicity can bereduced by transferring murine CDR sequences into a human antibodyframework (reshaping/CDR grafting/humanization), as described for atherapeutic antibody by Riechmann et al., Nature, 332: 323-327 (1988).Another method is referred to as “resurfacing” or “veneering”, startingwith the rodent variable light and heavy domains, onlysurface-accessible framework amino acids are altered to human ones,while the CDR and buried amino acids remain from the parental rodentantibody (Roguska et al., Protein Eng., 9(10): 895-904 (1996)). Inanother type of humanization, instead of grafting the entire CDRs, onetechnique grafts only the “specificity-determining regions” (SDRs),defined as the subset of CDR residues that are involved in binding ofthe antibody to its target (Kashmiri et al., Methods, 36(1): 25-34(2005)). This necessitates identification of the SDRs either throughanalysis of available three-dimensional structures of antibody-targetcomplexes or mutational analysis of the antibody CDR residues todetermine which interact with the target. Alternatively, fully humanantibodies may have reduced immunogenicity compared to murine, chimeric,or humanized antibodies.

Another approach to reduce the immunogenicity of therapeutic antibodiesis the elimination of certain specific sequences that are predicted tobe immunogenic. In one approach, after a first generation biologic hasbeen tested in humans and found to be unacceptably immunogenic, theB-cell epitopes can be mapped and then altered to avoid immunedetection. Another approach uses methods to predict and remove potentialT-cell epitopes. Computational methods have been developed to scan andto identify the peptide sequences of biologic therapeutics with thepotential to bind to MHC proteins (Desmet et al., Proteins, 58: 53-69(2005)). Alternatively a human dendritic cell-based method can be usedto identify CD4⁺ T-cell epitopes in potential protein allergens(Stickler et al., J. Immunother., 23: 654-660 (2000); Morrison andSchlom, Important Adv. Oncol. (1990) pp. 3-18; Riechmann et al.“Reshaping human antibodies for therapy,” Nature. 332: 323-327 (1988);Roguska et al., “A comparison of two murine mAbs humanized byCDR-grafting and variable domain resurfacing,” Protein Eng., 9: 895-904(1996); Kashmiri et al., “SDR grafting—a new approach to antibodyhumanization,” Methods, 36(1): 25-34 (2005); Desmet et al., “Anchorprofiles of HLA-specific peptides: analysis by a novel affinity scoringmethod and experimental validation,” Proteins, 58: 53-69 (2005);Stickler et al., “CD4+ T-cell epitope determination using unexposedhuman donor peripheral blood mononuclear cells,” J. Immunother., 23:654-660 (2000)).

B7. In Vivo Efficacy

To generate a DVD-Ig molecule with desired in vivo efficacy, it isimportant to generate and select mAbs with similarly desired in vivoefficacy when given in combination. However, in some instances theDVD-Ig may exhibit in vivo efficacy that cannot be achieved with thecombination of two separate mAbs. For instance, a DVD-Ig may bring twotargets in close proximity leading to an activity that cannot beachieved with the combination of two separate mAbs. Additional desirablebiological functions are described herein in section B3. Parentantibodies with characteristics desirable in the DVD-Ig molecule may beselected based on factors such as pharmacokinetic half-life (t½); tissuedistribution; soluble versus cell surface targets; and targetconcentration—soluble/density—surface.

B8. In Vivo Tissue Distribution

To generate a DVD-Ig molecule with desired in vivo tissue distribution,in an embodiment, parent mAbs with similar desired in vivo tissuedistribution profile must be selected. Alternatively, based on themechanism of the dual-specific targeting strategy, it may at other timesnot be required to select parent mAbs with the similarly desired in vivotissue distribution when given in combination. For instance, in the caseof a DVD-Ig in which one binding component targets the DVD-Ig to aspecific site thereby bringing the second binding component to the sametarget site. For example, one binding specificity of a DVD-Ig couldtarget pancreas (islet cells) and the other specificity could bring GLP1to the pancreas to induce insulin.

B9. Isotype

To generate a DVD-Ig molecule with desired properties including, but notlimited to, isotype, effector functions, and the circulating half-life,parent mAbs are selected that possess appropriate Fc-effector functionsdepending on the therapeutic utility and the desired therapeuticend-point. There are five main heavy chain classes or isotypes, some ofwhich have several sub-types and these determine the effector functionsof an antibody molecule. These effector functions reside in the hingeregion, CH₂, and CH3 domains of the antibody molecule. However, residuesin other parts of an antibody molecule may have effects on effectorfunctions as well. The hinge region Fc-effector functions include: (i)antibody-dependent cellular cytotoxicity (ADCC), (ii) complement (Clq)binding, activation, and complement-dependent cytotoxicity (CDC), (iii)phagocytosis/clearance of antigen-antibody complexes, and (iv) cytokinerelease in some instances. These Fc-effector functions of an antibodymolecule are mediated through the interaction of the Fc-region with aset of class-specific cell surface receptors. Antibodies of the IgG1isotype are most active while IgG2 and IgG4 having minimal or noeffector functions. The effector functions of the IgG antibodies aremediated through interactions with three structurally homologouscellular Fc receptor types (and sub-types) (FcgRl, FcgR11, and FcgRIII).These effector functions of an IgG1 can be eliminated by mutatingspecific amino acid residues in the lower hinge region (e.g., L234A,L235A) that are required for FcgR and Clq binding. Amino acid residuesin the Fc region, in particular the CH2-CH3 domains, also determine thecirculating half-life of the antibody molecule. This Fc function ismediated through the binding of the Fc-region to the neonatal Fcreceptor (FcRn), which is responsible for recycling of antibodymolecules from the acidic lysosomes back to the general circulation.

Whether a mAb should have an active or an inactive isotype will dependon the desired therapeutic end-point for an antibody. Some examples ofusage of isotypes and desired therapeutic outcome are listed below:

1. If the desired end-point is functional neutralization of a solublecytokine then an inactive isotype may be used;

2. If the desired out-come is clearance of a pathological protein anactive isotype may be used;

3. If the desired out-come is clearance of protein aggregates an activeisotype may be used;

4. If the desired outcome is to antagonize a surface receptor aninactive isotype is used (Tysabri, IgG4; OKT3®, mutated IgG1);

5. If the desired outcome is to eliminate target cells an active isotypeis used (Herceptin, IgG1 (and with enhanced effector functions); and

6. If the desired outcome is to clear proteins from circulation withoutentering the CNS an IgM isotype may be used (e.g., clearing circulatingAb peptide species).

The Fc effector functions of a parental mAb can be determined by variousin vitro methods well known in the art.

As discussed, the selection of isotype, and thereby the effectorfunctions will depend upon the desired therapeutic end-point. In caseswhere simple neutralization of a circulating target is desired, forexample blocking receptor-ligand interactions, the effector functionsmay not be required. In such instances, isotypes or mutations in theFc-region of an antibody that eliminate effector functions aredesirable. In other instances where elimination of target cells is thetherapeutic end-point, for example elimination of tumor cells, isotypesor mutations or de-fucosylation in the Fc-region that enhance effectorfunctions are desirable (Presta, L. G., Adv. Drug Del. Rev., 58: 640-656(2006); Satoh et al., Expert Opin. Biol. Ther., 6: 1161-1173 (2006).Similarly, depending up on the therapeutic utility, the circulatinghalf-life of an antibody molecule can be reduced/prolonged by modulatingantibody-FcRn interactions by introducing specific mutations in the Fcregion (Dall'Acqua et al., J. Biol. Chem., 281: 23514-23524 (2006);Petkova et al., Int. Immunol., 18: 1759-1769 (2006); Vaccaro et al.,Proc. Natl. Acad. Sci. USA, 103: 18709-18714 (2006).

The published information on the various residues that influence thedifferent effector functions of a normal therapeutic mAb may need to beconfirmed for a DVD-Ig. It may be possible that in a DVD-Ig formatadditional (different) Fc-region residues, other than those identifiedfor the modulation of monoclonal antibody effector functions, may beimportant.

Overall, the decision as to which Fc-effector functions (isotype) willbe critical in the final DVD-Ig format will depend up on the diseaseindication, therapeutic target, desired therapeutic end-point, andsafety considerations. Listed below are exemplary appropriate heavychain and light chain constant regions including, but not limited to:IgG1—allotype: G1mz; IgG1 mutant—A234, A235; IgG2—allotype: G2m(n-);Kappa—Km3; and Lambda.

Fc Receptor and Clq Studies:

The possibility of unwanted antibody-dependent cell-mediatedcytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) byantibody complexing to any overexpressed target on cell membranes can beabrogated by (for example, L234A, L235A) hinge-region mutations. Thesesubstituted amino acids, present in the IgG1 hinge region of mAb, areexpected to result in diminished binding of mAb to human Fc receptors(but not FcRn), as FcgR binding is thought to occur within overlappingsites on the IgG1 hinge region. This feature of mAb may lead to animproved safety profile over antibodies containing a wild-type IgG.Binding of mAb to human Fc receptors can be determined by flow cytometryexperiments using cell lines (e.g., THP-1, K562) and an engineered CHOcell line that expresses FcgRIIb (or other FcgRs). Compared to IgG1control monoclonal antibodies, mAb show reduced binding to FcgRI andFcgRIIa whereas binding to FcgRIIb is unaffected. The binding andactivation of C1q by antigen/IgG immune complexes triggers the classicalcomplement cascade with consequent inflammatory and/or immunoregulatoryresponses. The C1q binding site on IgGs has been localized to residueswithin the IgG hinge region. C1q binding to increasing concentrations ofmAb was assessed by C1q ELISA. The results demonstrate that mAb isunable to bind to C1q, as expected when compared to the binding of awildtype control IgG1. Overall, the L234A, L235A hinge region mutationabolishes binding of mAb to FcgRI, FcgRIIa, and C1q, but does not impactthe interaction of mAb with FcgRIIb. These data suggest that in vivo mAbwith mutant Fc will interact normally with the inhibitory FcgRIIb butwill likely fail to interact with the activating FcgRI and FcgRIIareceptors or C1q.

Human FcRn Binding:

The neonatal receptor (FcRn) is responsible for transport of IgG acrossthe placenta and to control the catabolic half-life of the IgGmolecules. It might be desirable to increase the terminal half-life ofan antibody to improve efficacy, to reduce the dose or frequency ofadministration, or to improve localization to the target. Alternatively,it might be advantageous to do the converse that is, to decrease theterminal half-life of an antibody to reduce whole body exposure or toimprove the target-to-non-target binding ratios. Tailoring theinteraction between IgG and its salvage receptor, FcRn, offers a way toincrease or decrease the terminal half-life of IgG. Proteins in thecirculation, including IgG, are taken up in the fluid phase throughmicropinocytosis by certain cells, such as those of the vascularendothelia. IgG can bind FcRn in endosomes under slightly acidicconditions (pH 6.0-6.5) and can recycle to the cell surface, where it isreleased under almost neutral conditions (pH 7.0-7.4). Mapping of theFc-region-binding site on FcRn80, 16, 17 showed that two histidineresidues that are conserved across species, His310 and His435, areresponsible for the pH dependence of this interaction. Usingphage-display technology, a mouse Fc-region mutation that increasesbinding to FcRn and extends the half-life of mouse IgG was identified(see Ghetie et al., Nature Biotechnol., 15(7): 637-640 (1997)).Fc-region mutations that increase the binding affinity of human IgG forFcRn at pH 6.0, but not at pH 7.4, have also been identified (see,Dall'Acqua et al., J. Immunol., 169(9): 5171-5180 (2002)). Moreover, inone case, a similar pH-dependent increase in binding (up to 27-fold) wasalso observed for rhesus FcRn, and this resulted in a twofold increasein serum half-life in rhesus monkeys compared with the parent IgG (see,Hinton et al., J. Biol. Chem., 279(8): 6213-6216 (2004)). These findingsindicate that it is feasible to extend the plasma half-life of antibodytherapeutics by tailoring the interaction of the Fc region with FcRn.Conversely, Fc-region mutations that attenuate interaction with FcRn canreduce antibody half-life.

B.10. Pharmacokinetics (PK)

To generate a DVD-Ig molecule with desired pharmacokinetic profile, inan embodiment, parent mAbs with the similarly desired pharmacokineticprofile are selected. One consideration is that immunogenic response tomonoclonal antibodies (i.e., “HAHA”, human anti-human antibody response;“HACA”, human anti-chimeric antibody response) further complicates thepharmacokinetics of these therapeutic agents. In an embodiment,monoclonal antibodies with minimal or no immunogenicity are used forconstructing DVD-Ig molecules such that the resulting DVD-Igs will alsohave minimal or no immunogenicity. Some of the factors that determinethe PK of a mAb include, but are not limited to, intrinsic properties ofthe mAb (VH amino acid sequence); immunogenicity; FcRn binding and Fcfunctions.

The PK profile of selected parental monoclonal antibodies can be easilydetermined in rodents as the PK profile in rodents correlates well with(or closely predicts) the PK profile of monoclonal antibodies incynomolgus monkey and humans.

After the parental monoclonal antibodies with desired PK characteristics(and other desired functional properties as discussed herein) areselected, the DVD-Ig is constructed. As the DVD-Ig molecules contain twoantigen-binding domains from two parental monoclonal antibodies, the PKproperties of the DVD-Ig are assessed as well. Therefore, whiledetermining the PK properties of the DVD-Ig, PK assays may be employedthat determine the PK profile based on functionality of bothantigen-binding domains derived from the 2 parent monoclonal antibodies.The PK profile of a DVD-Ig can be determined. Additional factors thatmay impact the PK profile of DVD-Ig include the antigen-binding domain(CDR) orientation, linker size, and Fc/FcRn interactions. PKcharacteristics of parent antibodies can be evaluated by assessing thefollowing parameters: absorption, distribution, metabolism andexcretion.

Absorption:

To date, administration of therapeutic monoclonal antibodies is viaparenteral routes (e.g., intravenous [IV], subcutaneous [SC], orintramuscular [IM]). Absorption of a mAb into the systemic circulationfollowing either SC or IM administration from the interstitial space isprimarily through the lymphatic pathway. Saturable, presystemic,proteolytic degradation may result in variable absolute bioavailabilityfollowing extravascular administration. Usually, increases in absolutebioavailability with increasing doses of monoclonal antibodies may beobserved due to saturated proteolytic capacity at higher doses. Theabsorption process for a mAb is usually quite slow as the lymph fluiddrains slowly into the vascular system, and the duration of absorptionmay occur over hours to several days. The absolute bioavailability ofmonoclonal antibodies following SC administration generally ranges from50% to 100%. In the case of a transport-mediating structure at theblood-brain barrier (BBB) targeted by the DVD-Ig construct, circulationtimes in plasma may be reduced due to enhanced trans-cellular transportat the blood brain barrier (BBB) into the CNS compartment, where theDVD-Ig is liberated to enable interaction via its second antigenrecognition site.

Distribution:

Following IV administration, monoclonal antibodies usually follow abiphasic serum (or plasma) concentration-time profile, beginning with arapid distribution phase, followed by a slow elimination phase. Ingeneral, a biexponential pharmacokinetic model best describes this kindof pharmacokinetic profile. The volume of distribution in the centralcompartment (Vc) for a mAb is usually equal to or slightly larger thanthe plasma volume (2-3 liters). A distinct biphasic pattern in serum(plasma) concentration versus time profile may not be apparent withother parenteral routes of administration, such as IM or SC, because thedistribution phase of the serum (plasma) concentration-time curve ismasked by the long absorption portion. Many factors, includingphysicochemical properties, site-specific and target-oriented receptormediated uptake, binding capacity of tissue, and mAb dose can influencebiodistribution of a mAb. Some of these factors can contribute tononlinearity in biodistribution for a mAb.

Metabolism and Excretion:

Due to the molecular size, intact monoclonal antibodies are not excretedinto the urine via kidney. They are primarily inactivated by metabolism(e.g., catabolism). For IgG-based therapeutic monoclonal antibodies,half-lives typically range from hours or 1-2 days to over 20 days. Theelimination of a mAb can be affected by many factors, including, but notlimited to, affinity for the FcRn receptor, immunogenicity of the mAb,the degree of glycosylation of the mAb, the susceptibility for the mAbto proteolysis, and receptor-mediated elimination.

B.11. Tissue Cross-Reactivity Pattern on Human and Tox Species

Identical staining pattern suggests that potential human toxicity can beevaluated in tox species. Tox species are those animal in whichunrelated toxicity is studied.

The individual antibodies are selected to meet two criteria: (1) tissuestaining appropriate for the known expression of the antibody target and(2) similar staining pattern between human and tox species tissues fromthe same organ.

Criterion 1: Immunizations and/or antibody selections typically employrecombinant or synthesized antigens (proteins, carbohydrates or othermolecules). Binding to the natural counterpart and counterscreen againstunrelated antigens are often part of the screening funnel fortherapeutic antibodies. However, screening against a multitude ofantigens is often unpractical. Therefore, tissue cross-reactivitystudies with human tissues from all major organs serve to rule outunwanted binding of the antibody to any unrelated antigens.

Criterion 2: Comparative tissue cross reactivity studies with human andtox species tissues (cynomolgus monkey, dog, possibly rodents, andothers, the same 36 or 37 tissues being tested as in the human study)help to validate the selection of a tox species. In the typical tissuecross-reactivity studies on frozen tissue sections, therapeuticantibodies may demonstrate the expected binding to the known antigenand/or to a lesser degree binding to tissues based either on low levelinteractions (unspecific binding, low level binding to similar antigens,low level charge based interactions, etc.). In any case, the mostrelevant toxicology animal species is the one with the highest degree ofcoincidence of binding to human and animal tissue.

Tissue cross-reactivity studies follow the appropriate regulatoryguidelines including EC CPMP Guideline III/5271/94 “Production andquality control of mAbs” and the 1997 US FDA/CBER “Points to Consider inthe Manufacture and Testing of Monoclonal Antibody Products for HumanUse”. Cryosections (5 μm) of human tissues obtained at autopsy or biopsywere fixed and dried on object glass. The peroxidase staining of tissuesections are performed, using the avidin-biotin system. FDA's Guidance“Points to Consider in the Manufacture and Testing of MonoclonalAntibody Products for Human Use”. Relevant references include Clarke, J.(2004), Boon, L. (2002a), Boon, L. (2002b), Ryan, A. (1999).

Tissue-cross reactivity studies are often done in two stages, with thefirst stage including cryosections of 32 tissues (typically: AdrenalGland, Gastrointestinal Tract, Prostate, Bladder, Heart, SkeletalMuscle, Blood Cells, Kidney, Skin, Bone Marrow, Liver, Spinal Cord,Breast, Lung, Spleen, Cerebellum, Lymph Node, Testes, Cerebral Cortex,Ovary, Thymus, Colon, Pancreas, Thyroid, Endothelium, Parathyroid,Ureter, Eye, Pituitary, Uterus, Fallopian Tube and Placenta) from onehuman donor. In the second phase, a full cross reactivity study isperformed with up to 38 tissues (including adrenal, blood, blood vessel,bone marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart,kidney, large intestine, liver, lung, lymph node, breast mammary gland,ovary, oviduct, pancreas, parathyroid, peripheral nerve, pituitary,placenta, prostate, salivary gland, skin, small intestine, spinal cord,spleen, stomach, striated muscle, testis, thymus, thyroid, tonsil,ureter, urinary bladder, and uterus) from three unrelated adults.Studies are done typically at minimally two dose levels.

The therapeutic antibody (i.e., test article) and isotype matchedcontrol antibody may be biotinylated for avidin-biotin complex (ABC)detection; other detection methods may include tertiary antibodydetection for a FITC (or otherwise) labeled test article, orprecomplexing with a labeled anti-human IgG for an unlabeled testarticle.

Briefly, cryosections (about 5 μm) of human tissues obtained at autopsyor biopsy are fixed and dried on object glass. The peroxidase stainingof tissue sections is performed, using the avidin-biotin system. First(in case of a precomplexing detection system), the test article isincubated with the secondary biotinylated anti-human IgG and developedinto immune complex. The immune complex at the final concentrations of 2and 10 μg/mL of test article is added onto tissue sections on objectglass and then the tissue sections were reacted for 30 minutes with aavidin-biotin-peroxidase kit. Subsequently, DAB (3,3′-diaminobenzidine),a substrate for the peroxidase reaction, was applied for 4 minutes fortissue staining Antigen-Sepharose beads are used as positive controltissue sections.

Any specific staining is judged to be either an expected (e.g.,consistent with antigen expression) or unexpected reactivity based uponknown expression of the target antigen in question. Any staining judgedspecific is scored for intensity and frequency. Antigen or serumcompetition or blocking studies can assist further in determiningwhether observed staining is specific or nonspecific.

If two selected antibodies are found to meet the selectioncriteria—appropriate tissue staining, matching staining between humanand toxicology animal specific tissue—they can be selected for DVD-Iggeneration.

The tissue cross-reactivity study has to be repeated with the finalDVD-Ig construct, but while these studies follow the same protocol asoutline herein, they are more complex to evaluate because any bindingcan come from any of the two parent antibodies, and any unexplainedbinding needs to be confirmed with complex antigen competition studies.

It is readily apparent that the complex undertaking of tissuecross-reactivity studies with a multispecific molecule like a DVD-Ig isgreatly simplified if the two parental antibodies are selected for: (1)lack of unexpected tissue cross-reactivity findings and (2) appropriatesimilarity of tissue cross-reactivity findings between the correspondinghuman and toxicology animal species tissues.

B.12. Specificity and Selectivity

To generate a DVD-Ig molecule with desired specificity and selectivity,one needs to generate and select parent mAbs with the similarly desiredspecificity and selectivity profile.

Binding studies for specificity and selectivity with a DVD-Ig can becomplex due to the four or more binding sites, two each for eachantigen. Briefly, binding studies using ELISA, BIAcore, KinExA, or otherinteraction studies with a DVD-Ig need to monitor the binding of one,two, or more antigens to the DVD-Ig molecule. While BIAcore technologycan resolve the sequential, independent binding of multiple antigens,more traditional methods including ELISA or more modern techniques likeKinExA cannot. Therefore careful characterization of each parentantibody is critical. After each individual antibody has beencharacterized for specificity, confirmation of specificity retention ofthe individual binding sites in the DVD-Ig molecule is greatlysimplified.

It is readily apparent that the complex undertaking of determining thespecificity of a DVD-Ig is greatly simplified if the two parentalantibodies are selected for specificity prior to being combined into aDVD-Ig.

Antigen-antibody interaction studies can take many forms, including manyclassical protein protein interaction studies, including ELISA (enzymelinked immunosorbent assay), mass spectrometry, chemical cross linking,SEC with light scattering, equilibrium dialysis, gel permeation,ultrafiltration, gel chromatography, large-zone analytical SEC,micropreparative ultracentrifugation (sedimentation equilibrium),spectroscopic methods, titration microcalorimetry, sedimentationequilibrium (in analytical ultracentrifuge), sedimentation velocity (inanalytical centrifuge), surface plasmon resonance (including BIAcore).Relevant references include Current Protocols in Protein Science, Volume3, chapters 19 and 20, (Coligan et al., eds.) (John Wiley & Sons Inc.)and references included therein; and Current Protocols in Immunology,(Coligan et al., eds.) (John Wiley & Sons Inc.) and relevant referencesincluded therein.

Cytokine Release in Whole Blood:

The interaction of mAb with human blood cells can be investigated by acytokine release assay (Wing et al., Therapeutic Immunol., 2(4): 183-190(1995); Current Protocols in Pharmacology, (Enna et al., eds.) (JohnWiley & Sons Inc.); Madhusudan et al., Clin. Cancer Res., 10(19):6528-6534 (2004); Cox et. al., Methods, 38(4): 274-282 (2006); Choi etal., Eur. J. Immunol., 31(1): 94-106 (2001)). Briefly, variousconcentrations of mAb are incubated with human whole blood for 24 hours.The concentration tested should cover a wide range including finalconcentrations mimicking typical blood levels in patients (including butnot limited to 100 ng/ml-100 μg/ml). Following the incubation,supernatants and cell lysates are analyzed for the presence of IL-1Rα,TNF-α, IL-1b, IL-6 and IL-8. Cytokine concentration profiles generatedfor mAb are compared to profiles produced by a negative human IgGcontrol and a positive LPS or PHA control. The cytokine profiledisplayed by mAb from both cell supernatants and cell lysates arecompared to that using control human IgG. In an embodiment, themonoclonal antibody does not interact with human blood cells tospontaneously release inflammatory cytokines.

Cytokine release studies for a DVD-Ig are complex due to the four ormore binding sites, two each for each antigen. Briefly, cytokine releasestudies as described herein measure the effect of the whole DVD-Igmolecule on whole blood or other cell systems, but cannot resolve whichportion of the molecule causes cytokine release. Once cytokine releasehas been detected, the purity of the DVD-Ig preparation has to beascertained, because some co-purifying cellular components can causecytokine release on their own. If purity is not the issue, fragmentationof DVD-Ig (including but not limited to removal of Fc portion,separation of binding sites etc.), binding site mutagenesis or othermethods may need to be employed to deconvolute any observations. It isreadily apparent that this complex undertaking is greatly simplified ifthe two parental antibodies are selected for lack of cytokine releaseprior to being combined into a DVD-Ig.

B.13. Cross-Reactivity to Other Species for Toxicological Studies

In an embodiment, the individual antibodies selected with sufficientcross-reactivity to appropriate tox species, for example, cynomolgusmonkey. Parental antibodies need to bind to orthologous species target(i.e., cynomolgus monkey) and elicit appropriate response (modulation,neutralization, activation). In an embodiment, the cross-reactivity(affinity/potency) to orthologous species target should be within10-fold of the human target. In practice, the parental antibodies areevaluated for multiple species, including mouse, rat, dog, monkey (andother non-human primates), as well as disease model species (i.e., sheepfor asthma model). The acceptable cross-reactivity to tox species fromthe parental monoclonal antibodies allows future toxicology studies ofDVD-Ig in the same species. For that reason, the two parental monoclonalantibodies should have acceptable cross-reactivity for a common toxspecies therefore allowing toxicology studies of DVD-Ig in the samespecies.

Parent mAbs may be selected from various mAbs capable of bindingspecific targets and well known in the art. These include, but are notlimited to IL-1β, anti-TNF antibody (U.S. Pat. No. 6,258,562),anti-IL-12 and/or anti-IL-12p40 antibody (U.S. Pat. No. 6,914,128);anti-IL-18 antibody (US Publication No. 2005/0147610 A1), anti-05,anti-CBL, anti-CD147, anti-gp120, anti-VLA-4, anti-CD11a, anti-CD18,anti-VEGF, anti-CD40L, anti CD-40 (e.g., see PCT Publication No. WO2007/124299) anti-Id, anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR,anti-TGF-beta 2, anti-HGF, anti-cMet, anti DLL-4, anti-NPR1, anti-PLGF,anti-ErbB3, anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-F gp,anti-CD11/18, anti-CD14, anti-ICAM-3, anti-RON, anti CD-19, anti-CD80(e.g., see PCT Publication No. WO 2003/039486), anti-CD4, anti-CD3,anti-CD23, anti-beta2-integrin, anti-alpha4beta7, anti-CD52, anti-HLADR, anti-CD22 (see, e.g., U.S. Pat. No. 5,789,554), anti-CD20, anti-MIF,anti-CD64 (FcR), anti-TCR alpha beta, anti-CD2, anti-Hep B, anti-CA 125,anti-EpCAM, anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25,anti-CD33, anti-HLA, anti-IGF1,2, anti-IGFR, anti-VNRintegrin,anti-IL-1alpha, anti-IL-1beta, anti-IL-1 receptor, anti-IL-2 receptor,anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5 receptor, anti-IL-6,anti-IL-6R, RANKL, NGF, DKK, alphaVbeta3, anti-IL-8, anti-IL-9,anti-IL-13, anti-IL-13 receptor, and anti-IL-23; IL-23p19; (see, Presta,L. G., “Selection, design, and engineering of therapeutic antibodies,”J. Allergy Clin. Immunol., 116: 731-736 (2005) and at worldwide websitehttp://www.path.cam.ac.uk/˜mrc7/humanisation/antibodies.html).

Parent mAbs may also be selected from various therapeutic antibodiesapproved for use, in clinical trials, or in development for clinicaluse. Such therapeutic antibodies include, but are not limited to,rituximab (Rituxan®, IDEC/Genentech/Roche) (see for example U.S. Pat.No. 5,736,137), a chimeric anti-CD20 antibody approved to treatNon-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently beingdeveloped by Genmab, an anti-CD20 antibody described in U.S. Pat. No.5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics,Inc.), HumaLYM (Intracel), and PRO70769 (PCT Publication No. WO2004/056312 (PCT/US2003/040426) entitled “Immunoglobulin Variants andUses Thereof”), trastuzumab (Herceptin®, Genentech) (see for exampleU.S. Pat. No. 5,677,171), a humanized anti-Her2/neu antibody approved totreat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg®), currently beingdeveloped by Genentech; an anti-Her2 antibody described in U.S. Pat. No.4,753,894; cetuximab (Erbitux®, ImClone) (U.S. Pat. No. 4,943,533; PCTPublication No. WO 96/40210), a chimeric anti-EGFR antibody in clinicaltrials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883),currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S.Ser. No. 10/172,317, published as US 2003/0091561, now U.S. Pat. No.7,247,301), currently being developed by Genmab; 425, EMD55900,EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy etal., Arch. Biochem. Biophys., 252(2): 773-783 (1991)); ICR62 (Instituteof Cancer Research) PCT publication No. WO 95/20045; 549-560 (1987);Rodeck et al., J. Cell Biochem., 35(4):315-320 (1987); Kettleborough etal., Protein Eng., 4(7): Modjtahedi et al., J. Cell Biophys.,22(1-3):129-146 (1993); Modjtahedi et al., Br. J. Cancer, 67(2):247-253(1993); Modjtahedi et al., Br. J. Cancer, 73(2):228-235 (1996);Modjtahedi et al., Int. J. Cancer, 105(2):273-280 (2003)); TheraCIM hR3(YM Biosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S.Pat. No. 5,891,996; U.S. Pat. No. 6,506,883; Mateo et al.,Immunotechnology, 3(1):71-81 (1997)); mAb-806 (Ludwig Institute forCancer Research, Memorial Sloan-Kettering) (Jungbluth et al., Proc.Natl. Acad. Sci. USA., 100(2): 639-644 (2003)); KSB-102 (KS Biomedix);MR1-1 (IVAX, National Cancer Institute) (PCT Publication No. WO01/62931); and SC100 (Scancell) (PCT Publication No. WO 01/88138);alemtuzumab (Campath®, Millennium), a humanized mAb currently approvedfor treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3(Orthoclone OKT3®), an anti-CD3 antibody developed by OrthoBiotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin(Mylotarg®), an anti-CD33 (p67 protein) antibody developed byCelltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fc fusion developedby Biogen), abciximab (ReoPro®), developed by Centocor/Lilly,basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®),developed by Medimmune, infliximab (Remicade®), an anti-TNFalphaantibody developed by Centocor, adalimumab (Humira®), an anti-TNFalphaantibody developed by Abbott Laboratories, Humicade®, an anti-TNFalphaantibody developed by Celltech, golimumab (CNTO-148), a fully human TNFantibody developed by Centocor, etanercept (Enbrel®), an p75 TNFreceptor Fc fusion developed by Immunex/Amgen, lenercept, an p55TNFreceptor Fc fusion previously developed by Roche, ABX-CBL, an anti-CD147antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody beingdeveloped by Abgenix, ABX-MA1, an anti-MUC18 antibody being developed byAbgenix, Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 in development byAntisoma, Therex (R1550), an anti-MUC1 antibody being developed byAntisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, beingdeveloped by Antisoma, Thioplatin (AS1407) being developed by Antisoma,Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) andalpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, ananti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, ananti-lymphotoxin beta receptor (LTBR) antibody being developed byBiogen, CAT-152, an anti-TGF-β2 antibody being developed by CambridgeAntibody Technology, ABT 874 (J695), an anti-IL-12 p40 antibody beingdeveloped by Abbott Laboratories, CAT-192, an anti-TGFβ1 antibody beingdeveloped by Cambridge Antibody Technology and Genzyme, CAT-213, ananti-Eotaxinl antibody being developed by Cambridge Antibody Technology,LymphoStat-B® an anti-Blys antibody being developed by CambridgeAntibody Technology and Human Genome Sciences Inc., TRAIL-R1 mAb, ananti-TRAIL-R1 antibody being developed by Cambridge Antibody Technologyand Human Genome Sciences, Inc., Avastin® bevacizumab, rhuMAb-VEGF), ananti-VEGF antibody being developed by Genentech, an anti-HER receptorfamily antibody being developed by Genentech, Anti-Tissue Factor (ATF),an anti-Tissue Factor antibody being developed by Genentech, Xolair®(Omalizumab), an anti-IgE antibody being developed by Genentech,Raptiva® (Efalizumab), an anti-CD11a antibody being developed byGenentech and Xoma, MLN-02 Antibody (formerly LDP-02), being developedby Genentech and Millennium Pharmaceuticals, HuMax CD4, an anti-CD4antibody being developed by Genmab, HuMax-IL15, an anti-IL15 antibodybeing developed by Genmab and Amgen, HuMax-Inflam, being developed byGenmab and Medarex, HuMax-Cancer, an anti-Heparanase I antibody beingdeveloped by Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma,being developed by Genmab and Amgen, HuMax-TAC, being developed byGenmab, IDEC-131, and anti-CD40L antibody being developed by IDECPharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody beingdeveloped by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibody beingdeveloped by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 beingdeveloped by IDEC Pharmaceuticals, anti-macrophage migration factor(MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, ananti-idiotypic antibody being developed by ImClone, IMC-1C11, ananti-KDR antibody being developed by ImClone, DC101, an anti-flk-1antibody being developed by ImClone, anti-VE cadherin antibodies beingdeveloped by ImClone, CEA-Cide® (labetuzumab), an anti-carcinoembryonicantigen (CEA) antibody being developed by Immunomedics, LymphoCide®(Epratuzumab), an anti-CD22 antibody being developed by Immunomedics,AFP-Cide, being developed by Immunomedics, MyelomaCide, being developedby Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide,being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody beingdeveloped by Medarex, MDX-060, an anti-CD30 antibody being developed byMedarex, MDX-070 being developed by Medarex, MDX-018 being developed byMedarex, Osidem® (IDM-1), and anti-Her2 antibody being developed byMedarex and Immuno-Designed Molecules, HuMax®-CD4, an anti-CD4 antibodybeing developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibodybeing developed by Medarex and Genmab, CNTO 148, an anti-TNFα antibodybeing developed by Medarex and Centocor/Johnson & Johnson, CNTO 1275, ananti-cytokine antibody being developed by Centocor/Johnson & Johnson,MOR101 and MOR102, anti-intercellular adhesion molecule-1 (ICAM-1)(CD54) antibodies being developed by MorphoSys, MOR201, ananti-fibroblast growth factor receptor 3 (FGFR-3) antibody beingdeveloped by MorphoSys, Nuvion® (visilizumab), an anti-CD3 antibodybeing developed by Protein Design Labs, HuZAF®, an anti-gamma interferonantibody being developed by Protein Design Labs, Anti-α 5β1 Integrin,being developed by Protein Design Labs, anti-IL-12, being developed byProtein Design Labs, ING-1, an anti-Ep-CAM antibody being developed byXoma, Xolair® (Omalizumab) a humanized anti-IgE antibody developed byGenentech and Novartis, and MLN01, an anti-Beta2 integrin antibody beingdeveloped by Xoma. In another embodiment, the therapeutics includeKRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for CancerResearch); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (alpha V133integrin, Medimmune); volociximab (alpha Vβ1 integrin, Biogen/PDL);Human mAb 216 (B cell glycosolated epitope, NCl); BiTE MT103 (bispecificCD19×CD3, Medimmune); 4G7×H22 (Bispecific BcellxFcgammaR1, Medarex/MerckKGa); rM28 (Bispecific CD28×MAPG, European Patent No. EP 1 444 268);MDX447 (EMD 82633) (Bispecific CD64×EGFR, Medarex); Catumaxomab(removab) (Bispecific EpCAM×anti-CD3, Trion/Fres); Ertumaxomab(bispecific HER2/CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125,ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888(CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab(MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab);Rituximab (Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20,Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23,Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDLBiopharma); HeFi-1, CD30, NCD; MDX-060 (CD30, Medarex); MDX-1401 (CD30,Medarex); SGN-30 (CD30, Seattle Genentics); SGN-33 (Lintuzumab) (CD33,Seattle Genentics); Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40,Novartis); SGN-40 (CD40, Seattle Genentics); Campathlh (Alemtuzumab)(CD52, Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38,Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293 (TRC093/D93)(cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab(MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab (Ticilimumab,CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab) (DR4TRAIL-R1 agonist,Human Genome Science/Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab(DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab)(DR5TRAIL-R2 agonist, HGS); Cetuximab (Erbitux) (EGFR, ImClone);IMC-11F8, (EGFR, ImClone); Nimotuzumab (EGFR, YM Bio); Panitumumab(Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab);CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201)(Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam, Glaxo/Centocor);MORAb-003 (folate receptor a, Morphotech); KW-2871 (ganglioside GD3.Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb,Celldex); Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma);AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP 751871(IGF1-R, Pfizer); IMC-A12 (IGF1-R, ImClone); BIIB022 (IGF-1R, Biogen);Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor);Anti-KIR (1-7F9) (Killer cell Ig-like Receptor (KIR), Novo); Hu3S193(Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LTBR,Biogen); HuHMFG1 (MUC1, Antisoma/NCl); RAV12 (N-linked carbohydrateepitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP),University of California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538)(PD1, Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa,ImClone); bavituximab (phosphatidylserine, Peregrine); huJ591 (PSMA,Cornell Research Foundation); muJ591 (PSMA, Cornell ResearchFoundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab(Remicade) (TNFa, Centocor); A27.15 (transferrin receptor, SalkInstitute, INSERM, PCT Publication No. WO 2005/111082); E2.3(transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF,Genentech); HuMV833 (VEGF, Tsukuba Research Lab, PCT Publication No. WO2000/034337, University of Texas); IMC-18F1 (VEGFR1, ImClone); IMC-1121(VEGFR2, ImClone).

C. Construction of DVD-Ig™ Binding Proteins

A multivalent multispecific dual variable domain immunoglobulin(DVD-Ig™) binding protein is designed such that two different lightchain variable domains (VL) from two different parent monoclonalantibodies are linked in tandem directly or via a short linker byrecombinant DNA techniques, followed by the light chain constant domain.Similarly, the heavy chain comprises two different heavy chain variabledomains (VH) linked in tandem, followed by the constant domain CH1 andFc region.

The variable domains can be obtained using recombinant DNA techniquesfrom a parent antibody generated by any one of the methods describedherein. In an embodiment, the variable domain is a murine heavy or lightchain variable domain. In another embodiment, the variable domain is aCDR-grafted or a humanized variable heavy or light chain domain. In anembodiment, the variable domain is a human heavy or light chain variabledomain.

In one embodiment, the first and second variable domains are linkeddirectly to each other using recombinant DNA techniques. In anotherembodiment the variable domains are linked via a linker sequence. In anembodiment, two variable domains are linked. Three or more variabledomains may also be linked directly or via a linker sequence. Thevariable domains may bind the same antigen or may bind differentantigens. DVD-Ig molecules of the invention may include oneimmunoglobulin variable domain and one non-immunoglobulin variabledomain such as ligand binding domain of a receptor, active domain of anenzyme. DVD-Ig molecules may also comprise two or more non-Ig domains.

The linker sequence may be a single amino acid or a linker polypeptidecomprising two or more amino acid residues joined by peptide bonds. Inan embodiment, a linker sequence is selected from the group consistingof GGGGSG (SEQ ID NO:26), GGSGG (SEQ ID NO:27), GGGGSGGGGS (SEQ IDNO:28), GGSGGGGSG (SEQ ID NO:223), GGSGGGGSGS (SEQ ID NO:29),GGSGGGGSGGGGS (SEQ ID NO:30), GGGGSGGGGSGGGG (SEQ ID NO:31),GGGGSGGGGSGGGGS (SEQ ID NO:32), ASTKGP (SEQ ID NO:33), ASTKGPSVFPLAP(SEQ ID NO:34), TVAAP (SEQ ID NO:35), RTVAAP (SEQ ID NO:224),TVAAPSVFIFPP (SEQ ID NO:36), RTVAAPSVFIFPP (SEQ ID NO:225),AKTTPKLEEGEFSEAR (SEQ ID NO:37), AKTTPKLEEGEFSEARV (SEQ ID NO:38),AKTTPKLGG (SEQ ID NO:39), SAKTTPKLGG (SEQ ID NO:40), SAKTTP (SEQ IDNO:41), RADAAP (SEQ ID NO:42), RADAAPTVS (SEQ ID NO:43), RADAAAAGGPGS(SEQ ID NO:44), RADAAAAGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:45),SAKTTPKLEEGEFSEARV (SEQ ID NO:46), ADAAP (SEQ ID NO:47), ADAAPTVSIFPP(SEQ ID NO:48), QPKAAP (SEQ ID NO:49), QPKAAPSVTLFPP (SEQ ID NO:50),AKTTPP (SEQ ID NO:51), AKTTPPSVTPLAP (SEQ ID NO:52), AKTTAP (SEQ IDNO:53), AKTTAPSVYPLAP (SEQ ID NO:54), GENKVEYAPALMALS (SEQ ID NO:55),GPAKELTPLKEAKVS (SEQ ID NO:56), and GHEAAAVMQVQYPAS (SEQ ID NO:57). Thechoice of linker sequences is based on crystal structure analysis ofseveral Fab molecules. There is a natural flexible linkage between thevariable domain and the CH1/CL constant domain in Fab or antibodymolecular structure. This natural linkage comprises approximately 10-12amino acid residues, contributed by 4-6 residues from C-terminus of Vdomain and 4-6 residues from the N-terminus of CL/CH1 domain. DVD-Igsdescribed herein can be generated using N-terminal 5-6 amino acidresidues, or 11-12 amino acid residues, of CL or CH1 as linker in lightchain and heavy chain of DVD-Ig, respectively. The N-terminal residuesof CL or CH1 domains, particularly the first 5-6 amino acid residues,adopt a loop conformation without strong secondary structures, andtherefore can act as flexible linkers between the two variable domains.The N-terminal residues of CL or CH1 domains are natural extension ofthe variable domains, as they are part of the Ig sequences, andtherefore minimize to a large extent any immunogenicity potentiallyarising from the linkers and junctions.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains; the light chain linkers canbe from Cκ or Cλ; and the heavy chain linkers can be derived from CH₁ ofany isotypes, including Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ.Linker sequences may also be derived from other proteins such as Ig-likeproteins, (e.g., TCR, FcR, KIR); G/S based sequences; hingeregion-derived sequences; and other natural sequences from otherproteins.

In an embodiment a constant domain is linked to the two linked variabledomains using recombinant DNA techniques. In an embodiment, a sequencecomprising tandemly linked heavy chain variable domains is linked to aheavy chain constant domain and a sequence comprising tandemly linkedlight chain variable domains is linked to a light chain constant domain.In an embodiment, the constant domains are human heavy chain constantdomain and human light chain constant domain, respectively. In anembodiment, the DVD heavy chain is further linked to an Fc region. TheFc region may be a native sequence Fc region, or a variant Fc region. Inanother embodiment, the Fc region is a human Fc region. In anotherembodiment the Fc region includes Fc region from IgG1, IgG2, IgG3, IgG4,IgA, IgM, IgE, or IgD.

In a most preferred embodiment, two heavy chain DVD polypeptides and twolight chain DVD polypeptides are combined to form a DVD-Ig molecule.Detailed description of specific DVD-Ig molecules capable of bindingspecific target antigens, such as IL-1β, and methods of making the sameare provided in the Examples section below.

D. Production of DVD-Ig Binding Proteins

DVD-Ig binding proteins of the present invention may be produced by anyof a number of techniques known in the art including, for example,expression from host cells, wherein expression vector(s) encoding theDVD-Ig heavy and DVD-Ig light chains is (are) transfected into a hostcell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. Although it ispossible to express the DVD-Ig proteins of the invention in eitherprokaryotic or eukaryotic host cells, DVD-Ig proteins are expressed ineukaryotic cells, for example, mammalian host cells, because sucheukaryotic cells (and in particular mammalian cells) are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active DVD-Ig protein.

Exemplary mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982)), NS0myeloma cells, COS cells, SP2 and PER.C6 cells. When recombinantexpression vectors encoding DVD-Ig proteins are introduced intomammalian host cells, the DVD-Ig proteins are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe DVD-Ig proteins in the host cells or secretion of the DVD proteinsinto the culture medium in which the host cells are grown. DVD-Igproteins can be recovered from the culture medium using standard proteinpurification methods.

In an exemplary system for recombinant expression of DVD-Ig proteins ofthe invention, a recombinant expression vector encoding both the DVD-Igheavy chain and the DVD-Ig light chain is introduced into dhfr-CHO cellsby calcium phosphate-mediated transfection. Within the recombinantexpression vector, the DVD-Ig heavy and light chain genes are eachoperatively linked to CMV enhancer/AdMLP promoter regulatory elements todrive high levels of transcription of the genes. The recombinantexpression vector also carries a DHFR gene, which allows for selectionof CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the DVD-Ig heavy and lightchains and intact DVD-Ig protein is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the DVD-Ig proteinfrom the culture medium. Still further the invention provides a methodof synthesizing a DVD-Ig protein of the invention by culturing a hostcell of the invention in a suitable culture medium until a DVD-Igprotein of the invention is synthesized. The method can further compriseisolating the DVD-Ig protein from the culture medium.

An important feature of DVD-Ig is that it can be produced and purifiedin a similar way as a conventional antibody. The production of DVD-Igresults in a homogeneous, single major product with desireddual-specific activity, without any sequence modification of theconstant region or chemical modifications of any kind. Other previouslydescribed methods to generate “bi-specific”, “multi-specific”, and“multi-specific multivalent” full length binding proteins do not lead toa single primary product but instead lead to the intracellular orsecreted production of a mixture of assembled inactive, mono-specific,multi-specific, multivalent, full length binding proteins, andmultivalent full length binding proteins with combination of differentbinding sites. As an example, based on the design described by Millerand Presta (PCT Publication No. WO 2001/077342, there are 16 possiblecombinations of heavy and light chains. Consequently only 6.25% ofprotein is likely to be in the desired active form, and not as a singlemajor product or single primary product compared to the other 15possible combinations. Separation of the desired, fully active forms ofthe protein from inactive and partially active forms of the proteinusing standard chromatography techniques, typically used in large scalemanufacturing, is yet to be demonstrated.

Surprisingly, the design of the “dual-specific multivalent full lengthbinding proteins” of the present invention leads to a dual variabledomain light chain and a dual variable domain heavy chain which assembleprimarily to the desired “dual-specific multivalent full length bindingproteins”.

At least 50%, at least 75%, and at least 90% of the assembled, andexpressed DVD-Ig molecules are the desired dual-specific tetravalentprotein. This aspect of the invention particularly enhances thecommercial utility of the invention. Therefore, the present inventionincludes a method to express a dual variable domain light chain and adual variable domain heavy chain in a single cell leading to a singleprimary product of a “dual-specific tetravalent full length bindingprotein”.

The present invention provides a methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a “primary product” of a “dual-specific, tetravalent,full length binding protein”, where the “primary product” is more than50% of all assembled protein, comprising a dual variable domain lightchain and a dual variable domain heavy chain.

The present invention provides methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a single “primary product” of a “dual-specific,tetravalent, full length binding protein”, where the “primary product”is more than 75% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

The present invention provides methods of expressing a dual variabledomain light chain and a dual variable domain heavy chain in a singlecell leading to a single “primary product” of a “dual-specifictetravalent full length binding protein”, where the “primary product” ismore than 90% of all assembled protein, comprising a dual variabledomain light chain and a dual variable domain heavy chain.

6. Production of IL-1β Binding Proteins and Binding Protein-ProducingCell Lines

Preferably, IL-1β binding proteins, including anti-IL-1β antibodies, ofthe present invention exhibit a high capacity to reduce or to neutralizeIL-1β activity, e.g., as assessed by any one of several in vitro and invivo assays known in the art. Preferably, IL-1β binding proteins of thepresent invention, also exhibit a high capacity to reduce or toneutralize IL-1β activity

In preferred embodiments, a binding protein, or antigen-binding portionthereof, binds human IL-1β, wherein the binding protein, orantigen-binding portion thereof, dissociates from human IL-1β with ak_(off) rate constant of about 0.1 s⁻¹ or less, as determined by surfaceplasmon resonance, or which inhibits human IL-1β activity with an IC₅₀of about 1×10⁻⁶M or less. Alternatively, the binding protein, or anantigen-binding portion thereof, may dissociate from human IL-1β with ak_(off) rate constant of about 1×10⁻² s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit human IL-1β activity with anIC₅₀ of about 1×10⁻⁷M or less. Alternatively, the binding protein, or anantigen-binding portion thereof, may dissociate from human IL-1β with ak_(off) rate constant of about 1×10⁻³ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit human IL-1β with an IC₅₀ ofabout 1×10⁻⁸M or less. Alternatively, the binding protein, or anantigen-binding portion thereof, may dissociate from human IL-1β with ak_(off) rate constant of about 1×10⁻⁴ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit human IL-1β activity with anIC₅₀ of about 1×10⁻⁹M or less. Alternatively, the binding protein, or anantigen-binding portion thereof, may dissociate from human IL-1β with ak_(off) rate constant of about 1×10⁻⁵ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit human IL IL-1β activity withan IC₅₀ of about 1×10⁻¹° M or less. Alternatively, the binding protein,or an antigen-binding portion thereof, may dissociate from human IL-1βwith a k_(off) rate constant of about 1×10⁻⁵ s⁻¹ or less, as determinedby surface plasmon resonance, or may inhibit human IL-1β activity withan IC₅₀ of about 1×10⁻¹¹M or less.

In certain embodiments, the binding protein comprises a heavy chainconstant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgDconstant region. Preferably, the heavy chain constant region is an IgG1heavy chain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (Winter et al., U.S. Pat. Nos.5,648,260 and 5,624,821). The Fc portion of an antibody mediates severalimportant effector functions e.g., cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC) andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment at least one amino acid residue is replacedin the constant region of the antibody, for example the Fc region of theantibody, such that effector functions of the antibody are altered.

One embodiment provides a labeled binding protein wherein an antibody orantibody portion of the invention is derivatized or linked to anotherfunctional molecule (e.g., another peptide or protein). For example, alabeled binding protein of the invention can be derived by functionallylinking an antibody or antibody portion of the invention (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

Useful detectable agents with which a binding protein, such as anantibody or antibody portion of the invention may be derivatized includefluorescent compounds. Exemplary fluorescent detectable agents includefluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike. An antibody may also be derivatized with detectable enzymes, suchas alkaline phosphatase, horseradish peroxidase, glucose oxidase and thelike. When an antibody is derivatized with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce adetectable reaction product. For example, when the detectable agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

Another embodiment of the invention provides a crystallized bindingprotein. Preferably the invention relates to crystals of wholeanti-IL-1β antibodies and fragments thereof as disclosed herein, andformulations and compositions comprising such crystals. In oneembodiment the crystallized binding protein has a greater half-life invivo than the soluble counterpart of the binding protein. In anotherembodiment the binding protein retains biological activity aftercrystallization.

Crystallized binding protein of the invention may be produced accordingmethods known in the art and as disclosed in PCT Publication No. WO02/072636, incorporated herein by reference.

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergo further processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins.

Naturally occurring antibodies are glycoproteins with one or morecarbohydrate residues in the Fc domain, as well as the variable domain.Carbohydrate residues in the Fc domain have important effect on theeffector function of the Fc domain, with minimal effect on antigenbinding or half-life of the antibody (Jefferis, R., Biotechnol. Prog.,21: 11-16 (2005)). In contrast, glycosylation of the variable domain mayhave an effect on the antigen binding activity of the antibody.Glycosylation in the variable domain may have a negative effect onantibody binding affinity, likely due to steric hindrance (Co et al.,Mol. Immunol., 30: 1361-1367 (1993)), or result in increased affinityfor the antigen (Wallick et al., J. Exp. Med., 168:1099-1109 (1988);Wright et al., EMBO J., 10: 2717-2723 (1991)).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the binding protein has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies.Glycosylation site mutants that retain the biological activity but haveincreased or decreased binding activity are another object of thepresent invention.

In still another embodiment, the glycosylation of the antibody orantigen-binding portion of the invention is modified. For example, anaglycoslated antibody can be made (i.e., the antibody lacksglycosylation). Glycosylation can be altered to, for example, increasethe affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region glycosylation sites to thereby eliminateglycosylation at that site. Such aglycosylation may increase theaffinity of the antibody for antigen. Such an approach is described infurther detail in PCT Publication No. WO 2003/016466, and U.S. Pat. Nos.5,714,350 and 6,350,861.

Additionally or alternatively, a modified binding protein of theinvention can be made that has an altered type of glycosylation, such asa hypofucosylated antibody having reduced amounts of fucosyl residues(see, Kanda et al., J. Biotechnol., 130(3): 300-310 (2007)) or anantibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields et al., J. Biol. Chem., 277: 26733-26740 (2002); Umana et al.,“Engineered glycoforms of an antineuroblastoma IgG1 with optimizedantibody-dependent cellular cytotoxic activity,” Nat. Biotechnol., 17:176-180 (1999), as well as, European Publication No. EP 1 176 195; PCTPublication Nos. WO 03/035835 and WO 99/54342.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. Preferably the glycosylated bindingprotein comprises glycosyl residues such that the glycosylation patternis human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (US Publication Nos.2004/0018590 and 2002/0137134).

In addition to the binding proteins, the present invention is alsodirected to anti-idiotypic (anti-Id) antibodies specific for suchbinding proteins of the invention. An anti-Id antibody is an antibody,which recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal with the binding protein or a CDR containingregion thereof. The immunized animal will recognize, and respond to theidiotypic determinants of the immunizing antibody and produce an anti-Idantibody. It is readily apparent that it may be easier to generateanti-idiotypic antibodies to the two or more parent antibodiesincorporated into a DVD-Ig molecule; and confirm binding studies bymethods well recognized in the art (e.g., BIAcore, ELISA) to verify thatanti-idiotypic antibodies specific for the idiotype of each parentantibody also recognize the idiotype (e.g., antigen binding site) in thecontext of the DVD-Ig. The anti-idiotypic antibodies specific for eachof the two or more antigen binding sites of a DVD-Ig provide idealreagents to measure DVD-Ig concentrations of a human DVD-Ig in patientserum. For example, DVD-Ig concentration assays can be established usinga “sandwich assay ELISA format” with an antibody to a first antigenbinding region coated on the solid phase (e.g., BIAcore chip, ELISAplate, etc.), rinsed with rinsing buffer, incubation with a serumsample, another rinsing step, and ultimately incubation with anotheranti-idiotypic antibody to the other antigen binding site, itselflabeled with an enzyme for quantitation of the binding reaction. In anembodiment, for a DVD-Ig with more than two different binding sites,anti-idiotypic antibodies to the two outermost binding sites (mostdistal and proximal from the constant region) will not only help indetermining the DVD-Ig concentration in human serum but also documentthe integrity of the molecule in vivo. Each anti-Id antibody may also beused as an “immunogen” to induce an immune response in yet anotheranimal, producing a so-called anti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

7. Uses of IL-1β Binding Proteins

Given their ability to bind to human IL-1β, the IL-1β binding proteins,or antigen binding portions thereof, of the invention can be used todetect IL-1β (e.g., in a biological sample, such as serum or plasma),using a conventional immunoassay, such as an enzyme linked immunosorbentassays (ELISA), an radioimmunoassay (RIA) or tissueimmunohistochemistry. The invention provides a method for detectingIL-1β in a biological sample comprising contacting a biological samplewith a binding protein, or antigen binding portion, of the invention anddetecting either the binding protein (or antigen binding portion) boundto IL-1β or unbound binding protein (or binding portion), to therebydetect IL-1β in the biological sample. The binding protein is directlyor indirectly labeled with a detectable substance to facilitatedetection of the bound or unbound antibody. Suitable detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

Alternative to labeling the binding protein, human IL-1β can be assayedin biological fluids by a competition immunoassay utilizing rh IL-1βstandards labeled with a detectable substance and an unlabeled humanIL-1β binding protein. In this assay, the biological sample, the labeledrh IL-1β standards, and the human IL-1β binding protein are combined andthe amount of labeled rh IL-1β standard bound to the unlabeled antibodyis determined. The amount of human IL-1β in the biological sample isinversely proportional to the amount of labeled rh IL-1β standard boundto the IL-1β binding protein. Similarly, human IL-1β can also be assayedin biological fluids by a competition immunoassay utilizing rh IL-1βstandards labeled with a detectable substance and an unlabeled humanIL-1β binding protein.

The binding proteins and IL-1β binding portions of the inventionpreferably are capable of neutralizing human IL-1β activity both invitro and in vivo. Accordingly, such binding proteins and IL-1β bindingportions thereof of the invention can be used to inhibit human IL-1βactivity, e.g., in a cell culture containing human IL-1β, in humansubjects, or in other mammalian subjects having IL-1β with which anantibody of the invention cross-reacts. In one embodiment, the inventionprovides a method for inhibiting human IL-1β activity comprisingcontacting human IL-1β with an IL-1β binding protein or binding portionthereof of the invention such that human IL-1β activity is inhibited.For example, in a cell culture containing, or suspected of containinghuman IL-1β, an IL-1β binding protein or binding portion thereof of theinvention can be added to the culture medium to inhibit human IL-1βactivity in the culture.

In another embodiment, the invention provides a method for reducinghuman IL-1β activity in a subject, advantageously from a subjectsuffering from a disease or disorder in which IL-1β activity isdetrimental. The invention provides methods for reducing IL-1β activityin a subject suffering from such a disease or disorder, which methodcomprises administering to the subject an antibody or antibody portionof the invention such that IL-1β activity in the subject is reduced.Preferably, the IL-1β is human IL-1β and the subject is a human subject.Alternatively, the subject can be a mammal expressing an IL-1β to whichan antibody of the invention is capable of binding. Still further, thesubject can be a mammal into which IL-1β has been introduced (e.g., byadministration of IL-1β or by expression of an IL-1β transgene). AnIL-1β binding protein of the invention can be administered to a humansubject for therapeutic purposes. Moreover, a binding protein of theinvention can be administered to a non-human mammal expressing an IL-1βwith which the antibody is capable of binding for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration).

As used herein, the term “a disorder in which IL-1β activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of IL-1β in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which IL-1βactivity is detrimental is a disorder in which reduction of IL-1βactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of IL-1β in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofIL-1β in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-IL-1β antibody as describedabove. Non-limiting examples of disorders that can be treated with theantibodies of the invention include those disorders discussed in thesection below pertaining to pharmaceutical compositions of theantibodies of the invention.

The DVD-Igs of the invention may bind IL-1β alone or multiple antigens(e.g., human IL-1β and another non-IL-1β antigen). Thus, a DVD-Ig mayblock or reduce activity of hu IL-1β and the activity of another targetantigen. Such other target antigens may include soluble targets (e.g.,IL-1α) and cell surface receptor targets (e.g., VEGFR, EGFR).

Such other antigens include, but are not limited to, the targets listedin publically available databases, which databases include those thatare available on the worldwide web and incorporated herein by reference.These target databases include:

Therapeutic targets (http://xin.cz3.nus.edu.sg/group/cjttd/ttd.asp);

Cytokines and cytokine receptors (http://www.cytokinewebfacts.com/,http://www.copewithcytokines.de/cope.cgi, and

http://cmbi.bjmu.edu.cn/cmbidata/cgf/CGF_Database/cytokine.medic.kumamoto-u.ac.jp/CFC/indexR.html);

Chemokines(http://cytokine.medic.kumamoto-u.ac.jp/CFC/CK/Chemokine.html);

Chemokine receptors and GPCRs(http://csp.medic.kumamoto-u.ac.jp/CSP/Receptor.html,http://www.gper.org/7tm/);

Olfactory Receptors(http://senselab.med.yale.edu/senselab/ORDB/default.asp);

Receptors (http://www.iuphar-db.org/iuphar-rd/list/index.htm);

Cancer targets (http://cged.hgc.jp/cgi-bin/input.cgi);

Secreted proteins as potential antibody targets(http://spd.cbi.pku.edu.cn/);

Protein kinases (http://spd.cbi.pku.edu.cn/), and

Human CD markers(http://content.labvelocity.com/tools/6/1226/CD_table_final_locked.pdf)and (Zola et al., “CD molecules 2005: human cell differentiationmolecules,” Blood, 106: 3123-3126 (2005)).

DVD-Igs are useful as therapeutic agents to simultaneously block two ormore different targets, i.e., human IL-1β and one or more othernon-IL-1β target antigens to enhance efficacy/safety and/or increasepatient coverage. Such targets may include soluble targets (TNF) andcell surface receptor targets (VEGFR and EGFR).

Additionally, DVD-Igs of the invention can be employed fortissue-specific delivery (target a tissue marker and a disease mediatorfor enhanced local PK thus higher efficacy and/or lower toxicity),including intracellular delivery (targeting an internalizing receptorand a intracellular molecule), delivering to inside brain (targetingtransferrin receptor and a CNS disease mediator for crossing theblood-brain barrier). DVD-Ig can also serve as a carrier protein todeliver an antigen to a specific location via binding to anon-neutralizing epitope of that antigen and also to increase thehalf-life of the antigen. Furthermore, DVD-Ig can be designed to eitherbe physically linked to medical devices implanted into patients ortarget these medical devices (see Burke et al., “Zotarolimus elutingstents,” Adv. Drug Deliv. Rev., 58(3): 437-446 (2006); Hildebrand etal., “Surface coatings for biological activation and functionalizationof medical devices,” Surface and Coatings Technology, 200(22-23):6318-6324 (2006); Wu et al., “Drug/device combinations for local drugtherapies and infection prophylaxis,” Biomaterials, 27: 2450-2467(2006); Marques et al., “Mediation of the Cytokine Network in theImplantation of Orthopedic Devices,” Chapter 21, In BiodegradableSystems in Tissue Engineering and Regenerative Medicine, (Reis et al.,eds.) (CRC Press LLC, Boca Raton, 2005) pp. 377-397). Briefly, directingappropriate types of cell to the site of medical implant may promotehealing and restoring normal tissue function. Alternatively, inhibitionof mediators (including but not limited to cytokines), released upondevice implantation by a DVD-Ig coupled to or target to a device is alsoprovided. For example, Stents have been used for years in interventionalcardiology to clear blocked arteries and to improve the flow of blood tothe heart muscle. However, traditional bare metal stents have been knownto cause restenosis (re-narrowing of the artery in a treated area) insome patients and can lead to blood clots. Recently, an anti-CD34antibody coated stent has been described which reduced restenosis andprevents blood clots from occurring by capturing endothelial progenitorcells (EPC) circulating throughout the blood. Endothelial cells arecells that line blood vessels, allowing blood to flow smoothly. The EPCsadhere to the hard surface of the stent forming a smooth layer that notonly promotes healing but prevents restenosis and blood clots,complications previously associated with the use of stents (Aoki et al.,J. Am. Coll. Cardiol., 45(10): 1574-1579 (2005)). In addition toimproving outcomes for patients requiring stents, there are alsoimplications for patients requiring cardiovascular bypass surgery. Forexample, a prosthetic vascular conduit (artificial artery) coated withanti-EPC antibodies would eliminate the need to use arteries frompatients legs or arms for bypass surgery grafts. This would reducesurgery and anesthesia times, which in turn will reduce coronary surgerydeaths. DVD-Ig are designed in such a way that it binds to a cellsurface marker (such as CD34) as well as a protein (or an epitope of anykind, including but not limited to proteins, lipids and polysaccharides)that has been coated on the implanted device to facilitate the cellrecruitment. Such approaches can also be applied to other medicalimplants in general. Alternatively, DVD-Igs can be coated on medicaldevices and upon implantation and releasing all DVDs from the device (orany other need which may require additional fresh DVD-Ig, includingaging and denaturation of the already loaded DVD-Ig) the device could bereloaded by systemic administration of fresh DVD-Ig to the patient,where the DVD-Ig is designed to binds to a target of interest (acytokine, a cell surface marker (such as CD34) etc.) with one set ofbinding sites and to a target coated on the device (including a protein,an epitope of any kind, including but not limited to lipids,polysaccharides and polymers) with the other. This technology has theadvantage of extending the usefulness of coated implants.

A. Use of DVD-Igs in Various Diseases

DVD-Ig molecules of the invention are also useful as therapeuticmolecules to treat various diseases. Such DVD molecules may bind one ormore targets involved in a specific disease. Examples of such targets invarious diseases are described below.

Human Autoimmune and Inflammatory Response

In one aspect, a DVD-Ig binding protein of the invention is capable ofbinding human IL-1β and one or more antigens that have been implicatedin general autoimmune and inflammatory responses, including C5, CCL1(1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4),CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21(MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26,CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2),CXCL1, CXCL10 (IP-10), CXCL11 (1-TAC/IP-9), CXCL12 (SDF1), CXCL13,CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13,IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A,IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9,LTA, LTB, MIF, SCYE1 (endothelial Monocyte-activating cytokine), SPP1,TNF, TNFSF5, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R,ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R,TOLLIP, FADD, IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2,TRAF3, TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28,CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2,FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8,CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22,CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11,CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2,BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3,DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1,IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R,IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11,IL11RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15,IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA,LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1,TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144).

Asthma

Allergic asthma is characterized by the presence of eosinophilia, gobletcell metaplasia, epithelial cell alterations, airway hyperreactivity(AHR), and Th2 and Th1 cytokine expression, as well as elevated serumIgE levels. It is now widely accepted that airway inflammation is thekey factor underlying the pathogenesis of asthma, involving a complexinterplay of inflammatory cells such as T cells, B cells, eosinophils,mast cells and macrophages, and of their secreted mediators includingcytokines and chemokines. Corticosteroids are the most importantanti-inflammatory treatment for asthma today, however their mechanism ofaction is non-specific and safety concerns exist, especially in thejuvenile patient population. The development of more specific andtargeted therapies is therefore warranted.

Animal models such as OVA-induced asthma mouse model, where bothinflammation and AHR can be assessed, are known in the art and may beused to determine the ability of various DVD-Ig molecules to treatasthma. Animal models for studying asthma are disclosed in Coffman etal., J. Exp. Med., 201(12): 1875-1879 (2005); Lloyd et al., Adv.Immunol., 77: 263-295 (2001); Boyce et al., J. Exp. Med., 201(12):1869-1873 (2005); and Snibson et al., Clin. Exp. Allergy, 35(2): 146-152(2005). In addition to routine safety assessments of these target pairsspecific tests for the degree of immunosuppression may be warranted andhelpful in selecting the best target pairs (see Luster et al.,Toxicology, 92(1-3): 229-243 (1994); Descotes, J., Develop. Biol.Standard., 77: 99-102 (1992); Hart et al., J. Allergy Clin. Immunol.,108(2): 250-257 (2001)).

One aspect of the invention pertains to DVD-Ig molecules capable ofbinding IL-1β and one or more, for example two, targets selected fromthe group consisting of IL-4, IL-5, IL-8, IL-9, IL-13, IL-18, IL-5R(α),TNFSF4, IL-4R(α), interferon α, eotaxin, TSLP, PAR-2, PGD2, and IgE. Anembodiment includes a dual-specific anti-IL-1β/IL-1α DVD-Ig as atherapeutic agent beneficial for the treatment of asthma.

Rheumatoid Arthritis (RA)

Rheumatoid arthritis (RA), a systemic disease, is characterized by achronic inflammatory reaction in the synovium of joints and isassociated with degeneration of cartilage and erosion of juxta-articularbone. Many pro-inflammatory cytokines including TNF, chemokines, andgrowth factors are expressed in diseased joints. Systemic administrationof anti-TNF antibody or sTNFR fusion protein to mouse models of RA wasshown to be anti-inflammatory and joint protective. Various cytokines,included IL-1β have been implicated in RA. Clinical investigations inwhich the activity of TNF in RA patients was blocked with intravenouslyadministered infliximab (Harriman et al., “Summary of clinical trials inrheumatoid arthritis using infliximab, an anti-TNFalpha treatment,” Ann.Rheum. Dis., 58 (Suppl 1): I61-I64 (1999)), a chimeric anti-TNF mAb, hasprovided evidence that TNF regulates IL-6, IL-8, MCP-1, and VEGFproduction, recruitment of immune and inflammatory cells into joints,angiogenesis, and reduction of blood levels of matrixmetalloproteinases-1 and -3. A better understanding of the inflammatorypathway in rheumatoid arthritis has led to identification of othertherapeutic targets involved in rheumatoid arthritis. Promisingtreatments such as interleukin-6 antagonists (IL-6 receptor antibodyMRA, developed by Chugai, Roche (see Nishimoto et al., Arthritis Rheum.,50(6): 1761-1769 (2004)), CTLA4Ig (abatacept, Genovese et al.,“Abatacept for rheumatoid arthritis refractory to tumor necrosis factoralpha inhibition,” N. Engl. J. Med., 353: 1114-1123 (2005)), and anti-Bcell therapy (rituximab, Okamoto et al., “Rituximab for rheumatoidarthritis,” N. Engl. J. Med., 351: 1909 (2004)) have already been testedin randomized controlled trials over the past year. IL-1β and othercytokines, such as IL-15 and IL-18, have been identified as playing arole using RA animal models (therapeutic antibody HuMax-IL_(—)15, AMG714 see Baslund et al., Arthritis Rheum., 52(9): 2686-2692 (2005)).Dual-specific antibody therapy, combining anti-TNF and another mediator,such IL-1β, has great potential in enhancing clinical efficacy and/orpatient coverage. For example, blocking both TNF and VEGF canpotentially eradicate inflammation and angiogenesis, both of which areinvolved in pathophysiology of RA. A DVD-Ig binding protein capable ofblocking IL-1α and IL-1β is contemplated. In addition to routine safetyassessments of these target pairs, specific tests for the degree ofimmunosuppression may be warranted and helpful in selecting the besttarget pairs (see Luster et al., Toxicology, 92(1-3): 229-243 (1994);Descotes et al., Develop. Biol. Standard., 77: 99-102 (1992); Hart etal., J. Allergy Clin. Immunol., 108(2): 250-257 (2001)). Whether aDVD-Ig molecule will be useful for the treatment of rheumatoid arthritiscan be assessed using pre-clinical animal RA models such as thecollagen-induced arthritis mouse model. Other useful models are alsowell known in the art (see Brand, D. D., Comp. Med., 55:114-122 (2005)).Based on the cross-reactivity of the parental antibodies for human andmouse orthologues (e.g., reactivity for human and mouse TNF, human andmouse IL-15, etc.) validation studies in the mouse CIA model may beconducted with “matched surrogate antibody” derived DVD-Ig molecules.Briefly, a DVD-Ig based on two (or more) mouse target specificantibodies may be matched to the extent possible to the characteristicsof the parental human or humanized antibodies used for human DVD-Igconstruction (similar affinity, similar neutralization potency, similarhalf-life, etc.).

In an embodiment, a DVD-Ig of the invention that binds human IL-1β andanother non-IL-1β target may also be used to treat other diseases inwhich IL-1β plays. Such diseases include, but are not limited to SLE,multiple sclerosis (MS), sepsis, various neurological diseases, andcancers (including cervical, breast, gastric). A more extensive list ofdiseases and disorders in which IL-1β plays a role is also providedbelow.

An embodiment of the invention pertains to a DVD-Ig molecules capable ofbinding human IL-1β and one or more targets selected from the groupconsisting of IL-1α, TNFα, IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L,IL-18, VEGF, VLA-4, TNF13, CD45RB, CD200, IFN-γ, GM-CSF, FGF, C5, CD52,sclerostin, and CCR2.

Systemic Lupus Erythematosis (SLE)

The immunopathogenic hallmark of SLE is the polyclonal B cellactivation, which leads to hyperglobulinemia, autoantibody productionand immune complex formation. The fundamental abnormality appears to bethe failure of T cells to suppress the forbidden B cell clones due togeneralized T cell dysregulation. In addition, B and T-cell interactionis facilitated by several cytokines such as IL-10 as well asco-stimulatory molecules such as CD40 and CD40L, B7 and CD28 and CTLA-4,which initiate the second signal. These interactions together withimpaired phagocytic clearance of immune complexes and apoptoticmaterial, perpetuate the immune response with resultant tissue injury.

In one aspect, a DVD-Ig binding protein of the invention is capable ofbinding human IL-1β and one or more of the following antigens that havebeen implicated in SLE: B cell targeted therapies: CD-20, CD-22, CD-19,CD28, CD4, CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5,TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1,SLA2, CD81, IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK,GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA,KLF6, TNFRSF7, CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA,TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2,ITGA2, ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA, and NT5E.;co-stimulatory signals: CTLA4 or B7.1/B7.2; inhibition of B cellsurvival: BlyS, BAFF; Complement inactivation: C5; Cytokine modulation:the key principle is that the net biologic response in any tissue is theresult of a balance between local levels of proinflammatory oranti-inflammatory cytokines (see Sfikakis et al., Curr. Opin.Rheumatol., 17:550-557 (2005)). SLE is considered to be a Th-2 drivendisease with documented elevations in serum IL-4, IL-6, IL-10. DVD-Igscapable of binding one or more targets selected from the groupconsisting of IL-4, IL-6, IL-10, IFN-α, and TNF-α are also contemplated.Combination of targets discussed herein will enhance therapeuticefficacy for SLE which can be tested in a number of lupus preclinicalmodels (see, Peng S. L., Methods Mol. Med., 102: 227-272 (2004)). Basedon the cross-reactivity of the parental antibodies for human and mouseorthologues (e.g., reactivity for human and mouse CD₂O, human and mouseInterferon alpha etc.) validation studies in a mouse lupus model may beconducted with “matched surrogate antibody” derived DVD-Ig molecules;briefly, a DVD-Ig based two (or more) mouse target specific antibodiesmay be matched to the extent possible to the characteristics of theparental human or humanized antibodies used for human DVD-Igconstruction (similar affinity, similar neutralization potency, similarhalf-life etc.).

Multiple Sclerosis (MS)

Multiple sclerosis (MS) is a complex human autoimmune-type disease witha predominantly unknown etiology. Immunologic destruction of myelinbasic protein (MBP) throughout the nervous system is the major pathologyof multiple sclerosis. MS is a disease of complex pathologies, whichinvolves infiltration by CD4+ and CD8+ T cells and of response withinthe central nervous system. Expression in the CNS of cytokines, reactivenitrogen species and costimulator molecules have all been described inMS. Of major consideration are immunological mechanisms that contributeto the development of autoimmunity. In particular, antigen expression,cytokine and leukocyte interactions, and regulatory T-cells, which helpbalance/modulate other T-cells such as Th1 and Th2 cells, are importantareas for therapeutic target identification.

IL-12 is a proinflammatory cytokine that is produced by APC and promotesdifferentiation of Th1 effector cells. IL-12 is produced in thedeveloping lesions of patients with MS as well as in EAE-affectedanimals. Previously it was shown that interference in IL-12 pathwayseffectively prevents EAE in rodents, and that in vivo neutralization ofIL-12p40 using a anti-IL-12 mAb has beneficial effects in themyelin-induced EAE model in common marmosets.

TWEAK is a member of the TNF family, constitutively expressed in thecentral nervous system (CNS), with pro-inflammatory, proliferative orapoptotic effects depending upon cell types. Its receptor, Fn14, isexpressed in CNS by endothelial cells, reactive astrocytes and neurons.TWEAK and Fn14 mRNA expression increased in spinal cord duringexperimental autoimmune encephalomyelitis (EAE). Anti-TWEAK antibodytreatment in myelin oligodendrocyte glycoprotein (MOG) induced EAE inC57BL/6 mice resulted in a reduction of disease severity and leukocyteinfiltration when mice were treated after the priming phase.

One aspect of the invention pertains to DVD-Ig molecules capable ofbinding IL-1β and one or more, for example two, targets selected fromthe group consisting of IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18,VEGF, VLA-4, TNF, CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52,osteopontin, and CCR2. An embodiment includes a dual-specificanti-IL-1β/TWEAK DVD-Ig as a therapeutic agent beneficial for thetreatment of MS.

Several animal models for assessing the usefulness of the DVD-Igmolecules to treat MS are known in the art (see Steinman et al., TrendsImmunol., 26(11): 565-571 (2005); Lublin et al., Springer SeminImmunopathol., 8(3): 197-208 (1985); Genain et al., J. Mol. Med., 75(3):187-197 (1997); Tuohy et al., J. Exp. Med., 189(7): 1033-1042 (1999);Owens et al., Neurol. Clin., 13(1): 51-73 (1995); and 't Hart et al., J.Immunol., 175(7): 4761-4768 (2005)). Based on the cross-reactivity ofthe parental antibodies for human and animal species orthologues (e.g.,reactivity for human and mouse IL-β, human and mouse TWEAK etc.)validation studies in the mouse EAE model may be conducted with “matchedsurrogate antibody” derived DVD-Ig molecules; briefly, a DVD-Ig based onto (or more) mouse target specific antibodies may be matched to theextent possible to the characteristics of the parental human orhumanized antibodies used for human DVD-Ig construction (similaraffinity, similar neutralization potency, similar half-life etc.). Thesame concept applies to animal models in other non-rodent species, wherea “matched surrogate antibody” derived DVD-Ig would be selected for theanticipated pharmacology and possibly safety studies. In addition toroutine safety assessments of these target pairs specific tests for thedegree of immunosuppression may be warranted and helpful in selectingthe best target pairs (see Luster et al., Toxicology, 92(1-3): 229-243(1994); Descotes et al., Develop. Biol. Standard., 77: 99-102 (1992);Jones, R., “Rovelizumab-ICOS Corp,” IDrugs, 3(4):442-446 (2000)).

Sepsis

The pathophysiology of sepsis is initiated by the outer membranecomponents of both gram-negative organisms (lipopolysaccharide [LPS],lipid A, endotoxin) and gram-positive organisms (lipoteichoic acid,peptidoglycan). These outer membrane components are able to bind to theCD14 receptor on the surface of monocytes. By virtue of the recentlydescribed toll-like receptors, a signal is then transmitted to the cell,leading to the eventual production of the proinflammatory cytokinestumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1).Overwhelming inflammatory and immune responses are essential features ofseptic shock and play a central part in the pathogenesis of tissuedamage, multiple organ failure, and death induced by sepsis. Cytokines,especially tumor necrosis factor (TNF) and interleukin (IL-1), have beenshown to be critical mediators of septic shock. These cytokines have adirect toxic effect on tissues; they also activate phospholipase A2.These and other effects lead to increased concentrations ofplatelet-activating factor, promotion of nitric oxide synthase activity,promotion of tissue infiltration by neutrophils, and promotion ofneutrophil activity.

The treatment of sepsis and septic shock remains a clinical conundrum,and recent prospective trials with biological response modifiers (i.e.,anti-TNF, anti-MIF) aimed at the inflammatory response have shown onlymodest clinical benefit. Recently, interest has shifted toward therapiesaimed at reversing the accompanying periods of immune suppression.Studies in experimental animals and critically ill patients havedemonstrated that increased apoptosis of lymphoid organs and someparenchymal tissues contribute to this immune suppression, anergy, andorgan system dysfunction. During sepsis syndromes, lymphocyte apoptosiscan be triggered by the absence of IL-2 or by the release ofglucocorticoids, granzymes, or the so-called ‘death’ cytokines: tumornecrosis factor alpha or Fas ligand. Apoptosis proceeds viaauto-activation of cytosolic and/or mitochondrial caspases, which can beinfluenced by the pro- and anti-apoptotic members of the Bc1-2 family.In experimental animals, not only can treatment with inhibitors ofapoptosis prevent lymphoid cell apoptosis; it may also improve outcome.Although clinical trials with anti-apoptotic agents remain distant duein large part to technical difficulties associated with theiradministration and tissue targeting, inhibition of lymphocyte apoptosisrepresents an attractive therapeutic target for the septic patient.Likewise, a dual-specific agent targeting both inflammatory mediator anda apoptotic mediator, may have added benefit. One aspect of theinvention pertains to DVD-Igs capable of binding IL-1β and one or moretargets involved in sepsis selected from the group consisting TNF, IL-1,MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors,TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFκB1,PROC, TNFRSF1A, CSF3, CCR3, IL1RN, MIF, NFκB1, PTAFR, TLR2, TLR4, GPR44,HMOX1, HMG-B1, midkine, IRAK1, NFκB2, SERPINA1, SERPINE1, and TREM1. Theefficacy of such DVD-Igs for sepsis can be assessed in preclinicalanimal models known in the art (see, Buras et al., Nat. Rev. DrugDiscov., 4(10): 854-865 (2005); and Calandra et al., Nature Med.,6(2):164-170 (2000)).

Neurological Disorders and Neurodegenerative Diseases

Neurodegenerative diseases are either chronic in which case they areusually age-dependent or acute (e.g., stroke, traumatic brain injury,spinal cord injury, etc.). They are characterized by progressive loss ofneuronal functions (neuronal cell death, demyelination), loss ofmobility and loss of memory. Emerging knowledge of the mechanismsunderlying chronic neurodegenerative diseases (e.g., Alzheimer'sdisease, AD) show a complex etiology and a variety of factors have beenrecognized to contribute to their development and progression e.g., age,glycemic status, amyloid production and multimerization, accumulation ofadvanced glycation-end products (AGE) which bind to their receptor RAGE(receptor for AGE), increased brain oxidative stress, decreased cerebralblood flow, neuroinflammation including release of inflammatorycytokines and chemokines, neuronal dysfunction and microglialactivation. Thus these chronic neurodegenerative diseases represent acomplex interaction between multiple cell types and mediators. Treatmentstrategies for such diseases are limited and mostly constitute eitherblocking inflammatory processes with non-specific anti-inflammatoryagents (e.g., corticosteroids, COX inhibitors) or agents to preventneuron loss and/or synaptic functions. These treatments fail to stopdisease progression. Recent studies suggest that more targeted therapiessuch as antibodies to soluble Aβ peptide (including the Aβ oligomericforms) can not only help stop disease progression but may help maintainmemory as well. These preliminary observations suggest that specifictherapies targeting more than one disease mediator (e.g., Aβ and apro-inflammatory cytokine such as TNF) may provide even bettertherapeutic efficacy for chronic neurodegenerative diseases thanobserved with targeting a single disease mechanism (e.g., soluble Aβalone) (see Shepherd et al., Neuropathol. Appl. Neurobiol., 31: 503-511(2005); Nelson, R. B., Curr. Pharm. Des., 11: 3335-3352 (2005); Klein,W. L., Neurochem. Int., 41: 345-352 (2002); Janelsins et al., “Earlycorrelation of microglial activation with enhanced tumor necrosisfactor-alpha and monocyte chemoattractant protein-I expressionspecifically within the entorhinal cortex of triple transgenicAlzheimer's disease mice,” J. Neuroinflammation, 2(23): 1-12 (2005);Soloman, B., Curr. Alzheimer. Res., 1: 149-163 (2004); Klyubin et al.,Nature Med., 11:556-561 (2005); Arancio et al., EMBO J., 23: 4096-4105(2004); Bornemann et al., Am. J. Pathol., 158: 63-73 (2001); Deane etal., Nature Med., 9: 907-913 (2003); and Masliah et al., Neuron, 46:857-868 (2005)).

The DVD-Ig molecules of the invention can bind IL-1β and one or moretargets involved in chronic neurodegenerative diseases such asAlzheimer's. Such targets include, but are not limited to, any mediator,soluble or cell surface, implicated in AD pathogenesis, e.g., AGE (S100A, amphotericin), pro-inflammatory cytokines (e.g., IL-1), chemokines(e.g., MCP 1), molecules that inhibit nerve regeneration (e.g., Nogo,RGM A), molecules that enhance neurite growth (neurotrophins) andmolecules that can mediate transport at the blood brain barrier (e.g.,transferrin receptor, insulin receptor or RAGE). The efficacy of DVD-Igmolecules can be validated in pre-clinical animal models such as thetransgenic mice that over-express amyloid precursor protein or RAGE anddevelop Alzheimer's disease-like symptoms. In addition, DVD-Ig moleculescan be constructed and tested for efficacy in the animal models and thebest therapeutic DVD-Ig can be selected for testing in human patients.DVD-Ig molecules can also be employed for treatment of otherneurodegenerative diseases such as Parkinson's disease. Alpha-Synucleinis involved in Parkinson's pathology. A DVD-Ig capable of targetingIL-1β and LINGO-1, alpha-synuclein, and/or inflammatory mediators suchas TNF, IL-17, MCP-1 can prove effective therapy for Parkinson's diseaseand are contemplated in the invention.

Neuronal Regeneration and Spinal Cord Injury

Despite an increase in knowledge of the pathologic mechanisms, spinalcord injury (SCI) is still a devastating condition and represents amedical indication characterized by a high medical need. Most spinalcord injuries are contusion or compression injuries and the primaryinjury is usually followed by secondary injury mechanisms (inflammatorymediators e.g., cytokines and chemokines) that worsen the initial injuryand result in significant enlargement of the lesion area, sometimes morethan 10-fold. These primary and secondary mechanisms in SCI are verysimilar to those in brain injury caused by other means e.g., stroke. Nosatisfying treatment exists and high dose bolus injection ofmethylprednisolone (MP) is the only used therapy within a narrow timewindow of 8 hours post injury. This treatment, however, is only intendedto prevent secondary injury without causing any significant functionalrecovery. It is heavily criticized for the lack of unequivocal efficacyand severe adverse effects, like immunosuppression with subsequentinfections and severe histopathological muscle alterations. No otherdrugs, biologics or small molecules, stimulating the endogenousregenerative potential are approved, but promising treatment principlesand drug candidates have shown efficacy in animal models of SCI inrecent years. To a large extent the lack of functional recovery in humanSCI is caused by factors inhibiting neurite growth, at lesion sites, inscar tissue, in myelin as well as on injury-associated cells. Suchfactors are the myelin-associated proteins NogoA, OMgp and MAG, RGM A,the scar-associated CSPG (Chondroitin Sulfate Proteoglycans) andinhibitory factors on reactive astrocytes (some semaphorins andephrins). However, at the lesion site not only growth inhibitorymolecules are found but also neurite growth stimulating factors likeneurotrophins, laminin, L1 and others. This ensemble of neurite growthinhibitory and growth promoting molecules may explain that blockingsingle factors, like NogoA or RGM A, resulted in significant functionalrecovery in rodent SCI models, because a reduction of the inhibitoryinfluences could shift the balance from growth inhibition to growthpromotion. However, recoveries observed with blocking a single neuriteoutgrowth inhibitory molecule were not complete. To achieve faster andmore pronounced recoveries either blocking two neurite outgrowthinhibitory molecules, e.g., Nogo and RGM A, or blocking an neuriteoutgrowth inhibitory molecule and enhancing functions of a neuriteoutgrowth enhancing molecule, e.g., Nogo and neurotrophins, or blockinga neurite outgrowth inhibitory molecule, e.g., Nogo and apro-inflammatory molecule e.g., TNF, may be desirable (see McGee et al.,Trends Neurosci., 26: 193-198 (2003); Domeniconi et al., J. Neurol.Sci., 233: 43-47 (2005); Makwana et al., FEBS J., 272: 2628-2638 (2005);Dickson, B. J., Science, 298: 1959-1964 (2002); Teng et al., J.Neurosci. Res., 79: 273-278 (2005); Karnezis et al., Nature Neurosci.,7: 736 (2004); Xu et al., J. Neurochem., 91: 1018-1023 (2004)).

In one aspect, a DVD-Ig that binds human IL-1β may also bind one or bothof the target pairs such as NgR and RGM A; NogoA and RGM A; MAG and RGMA; OMgp and RGM A; RGM A and RGM B; CSPGs and RGM A; aggrecan, midkine,neurocan, versican, phosphacan, Te38 and TNF-α; Aβ globulomer-specificantibodies combined with antibodies promoting dendrite & axon sproutingare provided. Dendrite pathology is a very early sign of AD and it isknown that NOGO A restricts dendrite growth. One can combine such typeof ab with any of the SCI-candidate (myelin-proteins) Ab. Other DVD-Igtargets may include any combination of NgR-p75, NgR-Troy, NgR-Nogo66(Nogo), NgR-Lingo, Lingo-Troy, Lingo-p75, MAG or OMgp. Additionally,targets may also include any mediator, soluble or cell surface,implicated in inhibition of neurite, e.g., Nogo, OMgp, MAG, RGM A,semaphorins, ephrins, soluble Aβ, pro-inflammatory cytokines (e.g.,IL-1), chemokines (e.g., MIP 1a), molecules that inhibit nerveregeneration. The efficacy of anti-nogo/anti-RGM A or similar DVD-Igmolecules can be validated in pre-clinical animal models of spinal cordinjury. In addition, these DVD-Ig molecules can be constructed andtested for efficacy in the animal models and the best therapeutic DVD-Igcan be selected for testing in human patients. In addition, DVD-Igmolecules can be constructed that target two distinct ligand bindingsites on a single receptor e.g., Nogo receptor which binds three ligandNogo, OMgp, and MAG and RAGE that binds Aβ and S100 A. Furthermore,neurite outgrowth inhibitors e.g., nogo and nogo receptor, also play arole in preventing nerve regeneration in immunological diseases likemultiple sclerosis. Inhibition of nogo-nogo receptor interaction hasbeen shown to enhance recovery in animal models of multiple sclerosis.Therefore, DVD-Ig molecules that can block the function of one immunemediator, e.g., a cytokine like IL-12, and a neurite outgrowth inhibitormolecule, e.g., Nogo or RGM, may offer faster and greater efficacy thanblocking either an immune or a neurite outgrowth inhibitor moleculealone.

In general, antibodies do not cross the blood brain barrier (BBB) in anefficient and relevant manner. However, in certain neurologic diseases,e.g., stroke, traumatic brain injury, multiple sclerosis, etc., the BBBmay be compromised and allows for increased penetration of DVD-Igs andantibodies into the brain. In other neurological conditions, where BBBleakage is not occurring, one may employ the targeting of endogenoustransport systems, including carrier-mediated transporters such asglucose and amino acid carriers and receptor-mediatedtranscytosis-mediating cell structures/receptors at the vascularendothelium of the BBB, thus enabling trans-BBB transport of the DVD-Ig.Structures at the BBB enabling such transport include but are notlimited to the insulin receptor, transferrin receptor, LRP and RAGE. Inaddition, strategies enable the use of DVD-Igs also as shuttles totransport potential drugs into the CNS including low molecular weightdrugs, nanoparticles and nucleic acids (Coloma et al., Pharm. Res.,17(3):266-274 (2000); Boado et al., Bioconjug. Chem., 18(2):447-455(2007)).

Oncological Disorders

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (von Mehren et al., Ann. Rev. Med., 54: 343-369(2003)). Antibodies may exert antitumor effects by inducing apoptosis,redirected cytotoxicity, interfering with ligand-receptor interactions,or preventing the expression of proteins that are critical to theneoplastic phenotype. In addition, antibodies can target components ofthe tumor microenvironment, perturbing vital structures such as theformation of tumor-associated vasculature. Antibodies can also targetreceptors whose ligands are growth factors, such as the epidermal growthfactor receptor. The antibody thus inhibits natural ligands thatstimulate cell growth from binding to targeted tumor cells.Alternatively, antibodies may induce an anti-idiotype network,complement-mediated cytotoxicity, or antibody-dependent cellularcytotoxicity (ADCC). The use of dual-specific antibody that targets twoseparate tumor mediators will likely give additional benefit compared toa mono-specific therapy.

In another embodiment, a DVD-Ig that binds human IL-1β of the inventionmay also be capable of binding another target involved in oncologicaldiseases including, but not limited to: IGFR, IGF, VGFR1, PDGFRb,PDGFRa, IGF1,2, ERB3, CDCP, 1BSG2, ErbB3, CD52, CD₂O, CD19, CD3, CD4,CD8, BMP6, IL12A, IL1A, IL1B, 1L2, IL24, INHA, TNF, TNFSF10, BMP6, EGF,FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19,FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8,FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B, 1L2, INHA, TGFA, TGFB1, TGFB2,TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2,CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4,CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENOL, ERBB2, ESR1, ESR2, IGFBP3,IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL,TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1,IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3,NR2F6, NR4A3, ESR1, ESR2, NROB1, NROB2, NR1D2, NR1H2, NR1H4, NR112,NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2,NR5A1, NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOCl,BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10,FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2,IGFBP3, IGFBP6, IL12A, IL1A, IL1B, 1L2, IL24, INHA, INSL3, INSL4, KLK10,KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18,CDH19, CDH2O, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164,COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1,CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO₂, ENO₃,FASN, F1112584, F1125530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A,IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID,PR1, PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6,ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR,LAMAS, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3, BAI1,COL4A3, IL8, LAMAS, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4, PROK2,SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, IFNA1, IFNB1, IFNG, IL1B, 1L6, MDK, EDG1, EFNA1, EFNA3, EFNB2,EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2,TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B(p27Kipl), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin), CTSB(cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3,GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130),ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),NGFB (NGF), NGFR, NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5(maspin), SERPINE1 (PAI-1), TGFα, THBS1 (thrombospondin-1), TIE (Tie-1),TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7(claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin),GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin),KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type IIkeratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2(COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1(mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2,THBS4, and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4,phosphatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD55, CD38,CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL,VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2,NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1,PSMA, PSCA, SCD1, and CD59.

D. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising anantibody (including a DVD-Ig described herein), or antigen-bindingportion thereof, of the invention and a pharmaceutically acceptablecarrier. The pharmaceutical compositions comprising antibodies of theinvention are for use in, but not limited to, diagnosing, detecting, ormonitoring a disorder, in preventing, treating, managing, orameliorating of a disorder or one or more symptoms thereof, and/or inresearch. In a specific embodiment, a composition comprises one or moreantibodies of the invention. In another embodiment, the pharmaceuticalcomposition comprises one or more antibodies of the invention and one ormore prophylactic or therapeutic agents other than antibodies of theinvention for treating a disorder in which IL-1β activity isdetrimental. In an embodiment, the prophylactic or therapeutic agentsare known to be useful for or having been or currently being used in theprevention, treatment, management, or amelioration of a disorder or oneor more symptoms thereof. In accordance with these embodiments, thecomposition may further comprise of a carrier, diluent or excipient.

The antibodies and antibody portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antibody portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 262: 4429-4432(1987)), construction of a nucleic acid as part of a retroviral or othervector. Methods of administering a prophylactic or therapeutic agent ofthe invention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration, andmucosal administration (e.g., intranasal and oral routes). In addition,pulmonary administration can be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;5,855,913 and 5,290,540; and PCT Publication Nos. WO 92/19244, WO97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which isincorporated herein by reference their entireties. In one embodiment, anantibody or antibody portion of the invention, combination therapy, or acomposition of the invention is administered using Alkermes AIR®pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal, andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In an embodiment, specific binding of antibody-coupled carbon nanotubes(CNTs) to tumor cells in vitro, followed by their highly specificablation with near-infrared (NIR) light can be used to target tumorcells. For example, biotinylated polar lipids can be used to preparestable, biocompatible, noncytotoxic CNT dispersions that are thenattached to one or two different neutralite avidin-derivatized DVD-Igsdirected against one or more tumor antigens (e.g., CD22) (Chakravarty etal., Proc. Natl. Acad. Sci. USA, 105: 8697-8702 (2008)).

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more antibodies of theinvention antagonists is administered locally to the affected area to asubject to prevent, treat, manage, and/or ameliorate a disorder or asymptom thereof. In another embodiment, an effective amount of one ormore antibodies of the invention is administered locally to the affectedarea in combination with an effective amount of one or more therapies(e.g., one or more prophylactic or therapeutic agents) other than anantibody of the invention of a subject to prevent, treat, manage, and/orameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see Langer, supra; Sefton, M. V., CRC Crit. Rev. Biomed. Eng.,14: 201-240 (1987); Buchwald et al., Surgery, 88: 507-516 (1980); Saudeket al., N. Engl. J. Med., 321: 574-579 (1989)). In another embodiment,polymeric materials can be used to achieve controlled or sustainedrelease of the therapies of the invention (see, e.g., Goodson, J. M.,Chapter 6, In Medical Applications of Controlled Release, Vol. II,Applications and Evaluation, (Langer and Wise, eds.) (CRC Press, Inc.,Boca Raton, 1984) pp. 115-138; Langer and Peppas, J. Macromol. Sci. Rev.Macromol. Chem. Phys., C23(1): 61-126 (1983); see also Levy et al.,Science, 228:190-192 (1985); During et al., Ann. Neurol., 25:351-356(1989); Howard et al., J. Neurosurg., 71:105-112 (1989)); U.S. Pat. No.5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat.No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154;and PCT Publication No. WO 99/20253. Examples of polymers used insustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In anexemplary embodiment, the polymer used in a sustained releaseformulation is inert, free of leachable impurities, stable on storage,sterile, and biodegradable. In yet another embodiment, a controlled orsustained release system can be placed in proximity of the prophylacticor therapeutic target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Controlled release systems are discussed in the review by Langer(Science, 249:1527-1533 (1990)). Any technique known to one of skill inthe art can be used to produce sustained release formulations comprisingone or more therapeutic agents of the invention. See, e.g., U.S. Pat.No. 4,526,938, PCT Publication No. WO 91/05548, PCT Publication No. WO96/20698; Ning et al., “Intratumoral radioimmunotherapy of a human coloncancer xenograft using a sustained-release gel,” Radiotherapy Oncol.,39: 179-189 (1996); Song et al., “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA J. Pharm. Sci.Technol., 50: 372-377(1996); Cleek et al., “Biodegradable Polymeric Carriers for a bFGFAntibody for Cardiovascular Application,” Proceed. Intl. Symp. Control.Rel. Bioact. Mater., 24: 853-854 (1997); and Lam et al.,“Microencapsulation of Recombinant Humanized Monoclonal Antibody forLocal Delivery,” Proceed. Intl. Symp. Control Rel. Bioact. Mater., 24:759-760 (1997), each of which is incorporated herein by reference intheir entireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic®, DuPont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., Proc. Natl. Acad. Sci. USA,88: 1864-1868 (1991)). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic, such as lignocamne, to ease pain at thesite of the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity preferablygreater than water are typically employed. Suitable formulationsinclude, without limitation, solutions, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like, which are, ifdesired, sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, preferably in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as FREON®) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gelcaps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320;5,985,309; 5,934,272; 5,874,064; 5,855,913; and 5,290,540; and PCTPublication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, andWO 99/66903, each of which is incorporated herein by reference theirentireties. In a specific embodiment, an antibody of the invention,combination therapy, and/or composition of the invention is administeredusing Alkermes AIR® pulmonary drug delivery technology (Alkermes, Inc.,Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

The methods of the invention may additionally comprise of administrationof compositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachet indicating the quantity of activeagent. Where the mode of administration is infusion, composition can bedispensed with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the mode of administration is by injection,an ampoule of sterile water for injection or saline can be provided sothat the ingredients may be mixed prior to administration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents, or pharmaceutical compositions ofthe invention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C. and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,preferably within 5 days, within 72 hours, within 48 hours, within 24hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours,or within 1 hour after being reconstituted. In an alternativeembodiment, one or more of the prophylactic or therapeutic agents orpharmaceutical compositions of the invention is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the agent. Preferably, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, atleast 75 mg/ml or at least 100 mg/ml. The liquid form should be storedat between 2° C. and 8° C. in its original container.

The antibodies and antibody portions of the invention can beincorporated into a pharmaceutical composition suitable for parenteraladministration. Preferably, the antibody or antibody-portions will beprepared as an injectable solution containing 0.1-250 mg/ml antibody.The injectable solution can be composed of either a liquid orlyophilized dosage form in a flint or amber vial, ampoule or pre-filledsyringe. The buffer can be L-histidine (1-50 mM), optimally 5-10 mM, atpH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but arenot limited to, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate. Sodium chloride can be used to modify the toxicityof the solution at a concentration of 0-300 mM (optimally 150 mM for aliquid dosage form). Cryoprotectants can be included for a lyophilizeddosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Othersuitable cryoprotectants include trehalose and lactose. Bulking agentscan be included for a lyophilized dosage form, principally 1-10%mannitol (optimally 2-4%). Stabilizers can be used in both liquid andlyophilized dosage forms, principally 1-50 mM L-Methionine (optimally5-10 mM). Other suitable bulking agents include glycine, arginine, canbe included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%).Additional surfactants include but are not limited to polysorbate 20 andBRIJ surfactants. The pharmaceutical composition comprising an antibodyor antibody portion of the invention prepared as an injectable solutionfor parenteral administration, can further comprise an agent useful asan adjuvant, such as those used to increase the absorption, ordispersion of a therapeutic protein (e.g., antibody). A particularlyuseful adjuvant is hyaluronidase (such as Hylenex® recombinant humanhyaluronidase). Addition of hyaluronidase in the injectable solutionimproves human bioavailability following parenteral administration,particularly subcutaneous administration. It also allows for greaterinjection site volumes (i.e., greater than 1 ml) with less pain anddiscomfort, and minimum incidence of injection site reactions (see, PCTPublication No. WO 2004/078140 and US Publication No. 2006/104968).

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In an exemplaryembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The binding proteins of the present invention can be administered by avariety of methods known in the art, although for many therapeuticapplications, the preferred route/mode of administration is subcutaneousinjection, intravenous injection or infusion. As will be appreciated bythe skilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound may be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, (J. R. Robinson, ed.) (Marcel Dekker, Inc., New York, 1978).

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingdisorders in which IL-1β activity is detrimental. For example, ananti-human IL-1β antibody or antibody portion of the invention may becoformulated and/or coadministered with one or more additionalantibodies that bind other targets (e.g., antibodies that bind othercytokines or that bind cell surface molecules). Furthermore, one or moreantibodies of the invention may be used in combination with two or moreof the foregoing therapeutic agents. Such combination therapies mayadvantageously utilize lower dosages of the administered therapeuticagents, thus avoiding possible toxicities or complications associatedwith the various monotherapies.

In certain embodiments, an antibody to IL-1β or fragment thereof islinked to a half-life extending vehicle known in the art. Such vehiclesinclude, but are not limited to, the Fc domain, polyethylene glycol, anddextran. Such vehicles are described, e.g., in U.S. Ser. No. 09/428,082(now U.S. Pat. No. 6,660,843) which is hereby incorporated by referencefor any purpose.

In a specific embodiment, nucleic acid sequences comprising nucleotidesequences encoding an antibody of the invention or another prophylacticor therapeutic agent of the invention are administered to treat,prevent, manage, or ameliorate a disorder or one or more symptomsthereof by way of gene therapy. Gene therapy refers to therapy performedby the administration to a subject of an expressed or expressiblenucleic acid. In this embodiment of the invention, the nucleic acidsproduce their encoded antibody or prophylactic or therapeutic agent ofthe invention that mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., Clinical Pharm., 12: 488-505(1993); Wu et al., “Delivery systems for gene therapy,” Biotherapy, 3:87-95 (1991); Tolstoshev, P., Ann. Rev. Pharmacol. Toxicol., 32: 573-596(1993); Mulligan, R. C., Science, 260: 926-932 (1993); and Morgan andAnderson, “Human Gene Therapy,” Ann. Rev. Biochem., 62:191-217 (1993);Robinson, C., Trends Biotechnol., 11:155 (1993). Methods commonly knownin the art of recombinant DNA technology which can be used are describedin Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley &Sons, New York (1993); and Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, New York (1990).Detailed description of various methods of gene therapy are disclosed inUS Publication No. 2005/0042664 A1, which is incorporated herein byreference.

IL-1 family members (IL-1β and IL-1α) play a critical role in thepathology associated with a variety of disorders involving immune andinflammatory elements. An IL-1 binding protein described herein may beadministered to an individual to treat such disorders. In an embodiment,a disorder that may be treated by a method of the invention comprisingadministering to a subject an IL-1 binding protein described hereinincludes, but is not limited to, diabetes; uveitis; neuropathic pain;osteoarthritic pain; inflammatory pain; rheumatoid arthritis;osteoarthritis; juvenile chronic arthritis; septic arthritis; Lymearthritis; psoriatic arthritis; reactive arthritis; spondyloarthropathy;systemic lupus erythematosus (SLE); Crohn's disease; ulcerative colitis;inflammatory bowel disease; autoimmune diabetes; insulin dependentdiabetes mellitus; thyroiditis; asthma; allergic diseases; psoriasis;dermatitis; scleroderma; graft versus host disease; organ transplantrejection; acute immune disease associated with organ transplantation;chronic immune disease associated with organ transplantation;sarcoidosis; atherosclerosis; disseminated intravascular coagulation(DIC); Kawasaki's disease; Grave's disease; nephrotic syndrome; chronicfatigue syndrome; Wegener's granulomatosis; Henoch-Schoenlein purpurea;microscopic vasculitis of the kidneys; chronic active hepatitis;autoimmune uveitis; septic shock; toxic shock syndrome; sepsis syndrome;cachexia; infectious diseases; parasitic diseases; acute transversemyelitis; Huntington's chorea; Parkinson's disease; Alzheimer's disease;stroke; primary biliary cirrhosis; hemolytic anemia; malignancies; heartfailure; myocardial infarction; Addison's disease; sporadicpolyglandular deficiency type I; polyglandular deficiency type II(Schmidt's syndrome); acute respiratory distress syndrome (ARDS);alopecia; alopecia greata; seronegative arthropathy; arthropathy;Reiter's disease; psoriatic arthropathy; ulcerative colitic arthropathy;enteropathic synovitis; chlamydia; Yersinia and Salmonella associatedarthropathy; spondyloarthropathy; atheromatous disease/arteriosclerosis;atopic allergy; autoimmune bullous disease; pemphigus vulgaris;pemphigus foliaceus; pemphigoid; linear IgA disease; autoimmunehaemolytic anaemia; Coombs positive haemolytic anaemia; acquiredpernicious anaemia; juvenile pernicious anaemia; myalgicencephalitis/Royal Free disease; chronic mucocutaneous candidiasis;giant cell arteritis (GCA); primary sclerosing hepatitis; cryptogenicautoimmune hepatitis; acquired immunodeficiency syndrome (AIDS);acquired immunodeficiency related diseases; hepatitis B; hepatitis C;common varied immunodeficiency (common variable hypogammaglobulinaemia);dilated cardiomyopathy; female infertility; ovarian failure; prematureovarian failure; fibrotic lung disease; cryptogenic fibrosingalveolitis; post-inflammatory interstitial lung disease; interstitialpneumonitis; connective tissue disease associated interstitial lungdisease; mixed connective tissue disease associated lung disease;systemic sclerosis associated interstitial lung disease; rheumatoidarthritis associated interstitial lung disease; systemic lupuserythematosus associated lung disease; dermatomyositis/polymyositisassociated lung disease; Sjögren's disease associated lung disease;ankylosing spondylitis associated lung disease; vasculitic diffuse lungdisease; haemosiderosis associated lung disease; drug-inducedinterstitial lung disease; fibrosis; radiation fibrosis; bronchiolitisobliterans; chronic eosinophilic pneumonia; lymphocytic infiltrativelung disease; postinfectious interstitial lung disease; gouty arthritis;autoimmune hepatitis; type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis); type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis); autoimmune mediated hypoglycaemia; type B insulin resistancewith acanthosis nigricans; hypoparathyroidism; osteoarthrosis; primarysclerosing cholangitis; psoriasis type 1; psoriasis type 2; idiopathicleucopaenia; autoimmune neutropaenia; renal disease NOS;glomerulonephritides; microscopic vasculitis of the kidneys; Lymedisease; discoid lupus erythematosus; idiopathic male infertility;nitric oxide-associated male infertility; sperm autoimmunity; multiplesclerosis (all subtypes, including primary progressive, secondaryprogressive, relapsing remitting); sympathetic ophthalmia; pulmonaryhypertension secondary to connective tissue disease; Goodpasture'ssyndrome; pulmonary manifestation of polyarteritis nodosa; acuterheumatic fever; rheumatoid spondylitis; Still's disease; systemicsclerosis; Sjörgren's syndrome; Takayasu's disease/arteritis; autoimmunethrombocytopaenia (AITP); idiopathic thrombocytopaenia; autoimmunethyroid disease; hyperthyroidism; goitrous autoimmune hypothyroidism(Hashimoto's disease); atrophic autoimmune hypothyroidism; primarymyxoedema; phacogenic uveitis; primary vasculitis; vitiligo; acute liverdisease; chronic liver disease; alcoholic cirrhosis; alcohol-inducedliver injury; cholestasis; idiosyncratic liver disease; drug-inducedhepatitis; non-alcoholic steatohepatitis; allergy; group B Streptococci(GBS) infection; mental disorders (e.g., depression and schizophrenia);Th2 Type and Th1 Type mediated diseases; acute and chronic pain(different forms of pain); cancer (such as lung, breast, stomach,bladder, colon, pancreas, ovarian, prostate, and rectal cancer);hematopoietic malignancies; leukemia; lymphoma; abetalipoproteinemia;acrocyanosis; acute and chronic parasitic or infectious processes; acuteleukemia; acute lymphoblastic leukemia (ALL); T-cell ALL; FAB ALL; acutemyeloid leukemia (AML); acute or chronic bacterial infection; acutepancreatitis; acute renal failure; adenocarcinomas; atrial ectopicbeats; AIDS dementia complex; alcohol-induced hepatitis; allergicconjunctivitis; allergic contact dermatitis; allergic rhinitis;allograft rejection; alpha-1-antitrypsin deficiency; amyotrophic lateralsclerosis; anemia; angina pectoris; anterior horn cell degeneration;anti-CD3 therapy; antiphospholipid syndrome; anti-receptorhypersensitivity reactions; aortic and peripheral aneurysms; aorticdissection; arterial hypertension; arteriosclerosis; arteriovenousfistula; ataxia; atrial fibrillation (sustained or paroxysmal); atrialflutter; atrioventricular block; B cell lymphoma; bone graft rejection;bone marrow transplant (BMT) rejection; bundle branch block; Burkitt'slymphoma; burns; cardiac arrhythmias; cardiac stun syndrome; cardiactumors; cardiomyopathy; cardiopulmonary bypass inflammation response;cartilage transplant rejection; cerebellar cortical degenerations;cerebellar disorders; chaotic or multifocal atrial tachycardia;chemotherapy associated disorders; chronic myelocytic leukemia (CML);chronic alcoholism; chronic inflammatory pathologies; chroniclymphocytic leukemia (CLL); chronic obstructive pulmonary disease(COPD); chronic salicylate intoxication; colorectal carcinoma;congestive heart failure; conjunctivitis; contact dermatitis; corpulmonale; coronary artery disease; Creutzfeldt-Jakob disease; culturenegative sepsis; cystic fibrosis; cytokine therapy associated disorders;dementia pugilistica; demyelinating diseases; dengue hemorrhagic fever;dermatitis; dermatologic conditions; diabetes mellitus; diabeticarteriosclerotic disease; diffuse Lewy body disease; dilated congestivecardiomyopathy; disorders of the basal ganglia; Down's syndrome inmiddle age; drug-induced movement disorders induced by drugs which blockCNS dopamine receptors; drug sensitivity; eczema; encephalomyelitis;endocarditis; endocrinopathy; epiglottitis; Epstein-Barr virusinfection; erythromelalgia; extrapyramidal and cerebellar disorders;familial hemophagocytic lymphohistiocytosis; fetal thymus implantrejection; Friedreich's ataxia; functional peripheral arterialdisorders; fungal sepsis; gas gangrene; gastric ulcer; glomerularnephritis; graft rejection of any organ or tissue; gram negative sepsis;gram positive sepsis; granulomas due to intracellular organisms; hairycell leukemia; Hallervorden-Spatz disease; Hashimoto's thyroiditis; hayfever; heart transplant rejection; hemochromatosis; hemodialysis;hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura;hemorrhage; hepatitis A; His bundle arrhythmias; HIV infection/HIVneuropathy; Hodgkin's disease; hyperkinetic movement disorders;hypersensitivity reactions; hypersensitivity pneumonitis; hypertension;hypokinetic movement disorders; hypothalamic-pituitary-adrenal axisevaluation; idiopathic Addison's disease; idiopathic pulmonary fibrosis(IPF); antibody mediated cytotoxicity; asthenia; infantile spinalmuscular atrophy; inflammation of the aorta; influenza a; ionizingradiation exposure; iridocyclitis/uveitis/optic neuritis;ischemia-reperfusion injury; ischemic stroke; juvenile rheumatoidarthritis; juvenile spinal muscular atrophy; Kaposi's sarcoma; kidneytransplant rejection; legionella; leishmaniasis; leprosy; lesions of thecorticospinal system; lipedema; liver transplant rejection; lymphedema;malaria; malignant lymphoma; malignant histiocytosis; malignantmelanoma; meningitis; meningococcemia; metabolic syndrome migraineheadache; idiopathic migraine headache; mitochondrial multisystemdisorder; mixed connective tissue disease; monoclonal gammopathy;multiple myeloma; multiple systems degenerations (Menzel;Dejerine-Thomas; Shy-Drager; and Machado-Joseph); myasthenia gravis;mycobacterium avium intracellulare; mycobacterium tuberculosis;myelodysplastic syndrome; myocardial infarction; myocardial ischemicdisorders; nasopharyngeal carcinoma; neonatal chronic lung disease;nephritis; nephrosis; neurodegenerative diseases; neurogenic I muscularatrophies; neutropenic fever; non-Hodgkin's lymphoma; occlusion of theabdominal aorta and its branches; occlusive arterial disorders; OKT3®therapy; orchitis/epididymitis; orchitis/vasectomy reversal procedures;organomegaly; osteoporosis; pancreas transplant rejection; pancreaticcarcinoma; paraneoplastic syndrome/hypercalcemia of malignancy;parathyroid transplant rejection; pelvic inflammatory disease; perennialrhinitis; pericardial disease; peripheral atherosclerotic disease;peripheral vascular disorders; peritonitis; pernicious anemia;pneumocystis carinii pneumonia; pneumonia; POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome); post perfusion syndrome; post pump syndrome;post-MI cardiotomy syndrome; preeclampsia; progressive supranucleopalsy; primary pulmonary hypertension; radiation therapy; Raynaud'sphenomenon; Raynaud's disease; Refsum's disease; regular narrow QRStachycardia; renovascular hypertension; reperfusion injury; restrictivecardiomyopathy; sarcomas; senile chorea; senile dementia of Lewy bodytype; seronegative arthropathies; shock; sickle cell anemia; skinallograft rejection; skin changes syndrome; small bowel transplantrejection; solid tumors; specific arrhythmias; spinal ataxia;spinocerebellar degenerations; streptococcal myositis; structurallesions of the cerebellum; subacute sclerosing panencephalitis; syncope;syphilis of the cardiovascular system; systemic anaphylaxis; systemicinflammatory response syndrome; systemic onset juvenile rheumatoidarthritis; telangiectasia; thromboangitis obliterans; thrombocytopenia;toxicity; transplants; trauma/hemorrhage; type III hypersensitivityreactions; type IV hypersensitivity; unstable angina; uremia; urosepsis;urticaria; valvular heart diseases; varicose veins; vasculitis; venousdiseases; venous thrombosis; ventricular fibrillation; viral and fungalinfections; viral encephalitis/aseptic meningitis; viral-associatedhemophagocytic syndrome; Wernicke-Korsakoff syndrome; Wilson's disease;xenograft rejection of any organ or tissue; acute coronary syndromes;acute idiopathic polyneuritis; acute inflammatory demyelinatingpolyradiculoneuropathy; acute ischemia; adult Still's disease; alopeciagreata; anaphylaxis; anti-phospholipid antibody syndrome; aplasticanemia; arteriosclerosis; atopic eczema; atopic dermatitis; autoimmunedermatitis; autoimmune disorder associated with Streptococcus infection;autoimmune enteropathy; autoimmune hearing loss; autoimmunelymphoproliferative syndrome (ALPS); autoimmune myocarditis; autoimmunepremature ovarian failure; blepharitis; bronchiectasis; bullouspemphigoid; cardiovascular disease; catastrophic antiphospholipidsyndrome; celiac disease; cervical spondylosis; chronic ischemia;cicatricial pemphigoid; clinically isolated syndrome (CIS) with risk formultiple sclerosis; conjunctivitis; childhood onset psychiatricdisorder; dacryocystitis; dermatomyositis; diabetic retinopathy; diskherniation; disk prolapse; drug induced immune hemolytic anemia;endocarditis; endometriosis; endophthalmitis; episcleritis; erythemamultiforme; erythema multiforme major; gestational pemphigoid;Guillain-Barré syndrome (GBS); hay fever; Hughes syndrome; idiopathicParkinson's disease; idiopathic interstitial pneumonia; IgE-mediatedallergy; immune hemolytic anemia; inclusion body myositis; infectiousocular inflammatory disease; inflammatory demyelinating disease;inflammatory heart disease; inflammatory kidney disease; iritis;keratitis; keratojunctivitis sicca; Kussmaul disease or Kussmaul-Meierdisease; Landry's paralysis; Langerhan's cell histiocytosis; livedoreticularis; macular degeneration; microscopic polyangiitis; MorbusBechterev; motor neuron disorders; mucous membrane pemphigoid; multipleorgan failure; myasthenia gravis; myelodysplastic syndrome; myocarditis;nerve root disorders; neuropathy; non-A non-B hepatitis; optic neuritis;osteolysis; pauciarticular JRA; peripheral artery occlusive disease(PAOD); peripheral vascular disease (PVD); peripheral artery; disease(PAD); phlebitis; polyarteritis nodosa (or periarteritis nodosa);polychondritis; polymyalgia rheumatica; poliosis; polyarticular JRA;polyendocrine deficiency syndrome; polymyositis; polymyalgia rheumatica(PMR); post-pump syndrome; primary Parkinsonism; secondary Parkinsonism;prostatitis; pure red cell aplasia; primary adrenal insufficiency;recurrent neuromyelitis optica; restenosis; rheumatic heart disease;SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis);secondary amyloidosis; shock lung; scleritis; sciatica; secondaryadrenal insufficiency; silicone associated connective tissue disease;Sneddon-Wilkinson dermatosis; spondylitis ankylosans; Stevens-Johnsonsyndrome (SJS); systemic inflammatory response syndrome; temporalarteritis; toxoplasmic retinitis; toxic epidermal necrolysis; transversemyelitis; TRAPS (tumor necrosis factor receptor type 1 (TNFR)-associatedperiodic syndrome); type B insulin resistance with acanthosis nigricans;type 1 allergic reaction; type II diabetes; urticaria; usualinterstitial pneumonia (UIP); vernal conjunctivitis; viral retinitis;Vogt-Koyanagi-Harada syndrome (VKH syndrome); wet macular degeneration;wound healing; Yersinia and Salmonella associated arthropathy.

The binding proteins of the invention can be used to treat humanssuffering from autoimmune diseases, in particular those associated withinflammation, rheumatoid arthritis (RA), osteoarthritis, psoriasis,multiple sclerosis (MS), and other autoimmune diseases.

An antibody or antibody portion of the invention also can beadministered with one or more additional therapeutic agents useful inthe treatment of autoimmune and inflammatory diseases.

In an embodiment, diseases that can be treated or diagnosed with thecompositions and methods of the invention include, but are not limitedto, primary and metastatic cancers, including carcinomas of breast,colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach,pancreas, liver, gallbladder and bile ducts, small intestine, urinarytract (including kidney, bladder and urothelium), female genital tract(including cervix, uterus, and ovaries as well as choriocarcinoma andgestational trophoblastic disease), male genital tract (includingprostate, seminal vesicles, testes, and germ cell tumors), endocrineglands (including the thyroid, adrenal, and pituitary glands), and skin,as well as hemangiomas, melanomas, sarcomas (including those arisingfrom bone and soft tissues as well as Kaposi's sarcoma), tumors of thebrain, nerves, eyes, and meninges (including astrocytomas, gliomas,glioblastomas, retinoblastomas, neuromas, neuroblastomas, schwannomas,and meningiomas), solid tumors arising from hematopoietic malignanciessuch as leukemias, and lymphomas (both Hodgkin's and non-Hodgkin'slymphomas).

In another embodiment, an antibody of the invention or antigen bindingportion thereof is used to treat cancer or in the prevention ofmetastases from a tumor. Such treatment may involve administration ofthe antibody or antigen binding portion thereof alone or in combinationwith another therapeutic agent or treatment, such as radiotherapy and/ora chemotherapeutic agent.

The antibodies of the invention, or antigen binding portions thereof,may be combined with agents that include but are not limited to,antineoplastic agents, radiotherapy, chemotherapy such as DNA alkylatingagents, cisplatin, carboplatin, anti-tubulin agents, paclitaxel,docetaxel, taxol, doxorubicin, gemcitabine, gemzar, anthracyclines,adriamycin, topoisomerase I inhibitors, topoisomerase II inhibitors,5-fluorouracil (5-FU), leucovorin, irinotecan, receptor tyrosine kinaseinhibitors (e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g.,celecoxib), kinase inhibitors, and siRNAs.

A binding protein of the invention also can be administered with one ormore additional therapeutic agents useful in the treatment of variousdiseases.

Antibodies of the invention, or antigen binding portions thereof, can beused alone or in combination to treat such diseases. It should beunderstood that the antibodies of the invention or antigen bindingportion thereof can be used alone or in combination with an additionalagent, e.g., a therapeutic agent, said additional agent being selectedby the skilled artisan for its intended purpose. For example, theadditional agent can be a therapeutic agent art-recognized as beinguseful to treat the disease or condition being treated by the antibodyof the present invention. The additional agent also can be an agent thatimparts a beneficial attribute to the therapeutic composition, e.g., anagent that affects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents if the combination is such that the formedcomposition can perform its intended function.

Preferred combinations are non-steroidal anti-inflammatory drug(s) alsoreferred to as NSAIDS which include drugs like ibuprofen. Otherpreferred combinations are corticosteroids including prednisolone; thewell known side-effects of steroid use can be reduced or even eliminatedby tapering the steroid dose required when treating patients incombination with the anti-IL-1β antibodies of this invention.Non-limiting examples of therapeutic agents for rheumatoid arthritiswith which an antibody or antibody portion of the invention can becombined include, but are not limited to, the following: cytokinesuppressive anti-inflammatory drug(s) (CSAIDs); antibodies to orantagonists of other human cytokines or growth factors, for example,TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15, IL-16,IL-18, IL-21, interferons, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies ofthe invention, or antigen binding portions thereof, can be combined withantibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25,CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA ortheir ligands including CD154 (gp39 or CD40L).

Preferred combinations of therapeutic agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade; preferredexamples include TNF antagonists like chimeric, humanized or human TNFantibodies, D2E7, (PCT Publication No. WO 97/29131), CA2 (Remicade™) CDP571, and soluble p55 or p75 TNF receptors, derivatives, thereof,(p75TNFR1gG (Enbrel™) or p55TNFR1gG (Lenercept), and also TNFαconverting enzyme (TACE) inhibitors; similarly IL-1 inhibitors(Interleukin-1-converting enzyme inhibitors, IL-1RA etc.) may beeffective for the same reason. Other preferred combinations includeInterleukin 11. Yet another preferred combination are other key playersof the autoimmune response which may act parallel to, dependent on or inconcert with IL-1β function. Yet another preferred combination arenon-depleting anti-CD4 inhibitors. Yet other preferred combinationsinclude antagonists of the co-stimulatory pathway CD80 (B7.1) or CD86(B7.2) including antibodies, soluble receptors or antagonistic ligands.

The antibodies of the invention, or antigen binding portions thereof,may also be combined with agents, such as methotrexate, 6-MP,azathioprine sulphasalazine, mesalazine, olsalazinechloroquinine/hydroxychloroquine, pencillamine, aurothiomalate(intramuscular and oral), azathioprine, colchicine, corticosteroids(oral, inhaled and local injection), beta-2 adrenoreceptor agonists(salbutamol, terbutaline, salmeteral), xanthines (theophylline,aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium andoxitropium, cyclosporin, FK506, rapamycin, mycophenolate mofetil,leflunomide, NSAIDs, for example, ibuprofen, corticosteroids such asprednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signaling by proinflammatory cytokines such asTNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors),IL-1β converting enzyme inhibitors, TNFα converting enzyme (TACE)inhibitors, T-cell signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors and the derivatives p75TNFRIgG (Enbrel™ and p55TNFRIgG(Lenercept)), sIL-1R1, sIL-1R11, sIL-6R), antiinflammatory cytokines(e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ), celecoxib, folic acid,hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab, naproxen,valdecoxib, sulfasalazine, methylprednisolone, meloxicam,methylprednisolone acetate, gold sodium thiomalate, aspirin,triamcinolone acetonide, propoxyphene napsylate/apap, folate,nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium,oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenacsodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol hcl,salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen,alendronate sodium, prednisolone, morphine sulfate, lidocainehydrochloride, indomethacin, glucosamine sulf/chondroitin, amitriptylinehcl, sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,misoprostol, naproxen sodium, omeprazole, cyclophosphamide, rituximab,IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18, anti-IL15, BIRB-796,SCIO-469, VX-702, AMG-548, VX-740, Roflumilast, IC-485, CDC-801, andMesopram. Preferred combinations include methotrexate or leflunomide andin moderate or severe rheumatoid arthritis cases, cyclosporine.

Non-limiting additional agents which can also be used in combinationwith a binding protein to treat rheumatoid arthritis (RA) include, butare not limited to, the following: non-steroidal anti-inflammatorydrug(s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s)(CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFα antibody;Celltech/Bayer); cA2/infliximab (chimeric anti-TNFα antibody; Centocor);75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein;Immunex; see e.g., Moreland et al. (Abstract No. 813), Arthritis Rheum.,37: 5295 (1994); Baumgartner et al., J. Invest. Med., 44(3): 235A (March1996); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein;Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatizedanti-CD4 antibody; IDEC/SmithKline; see e.g., Kaine et al. (Abstract No.195), Arthritis Rheum., 38: 5185 (1995)); DAB 486-IL-2 and/or DAB389-IL-2 (IL-2 fusion proteins; Seragen; see e.g., Sewell et al.,Arthritis Rheum., 36(9): 1223-1233 (September 1993)); Anti-Tac(humanized anti-IL-2Ra; Protein Design Labs/Roche); IL-4(anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4;IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1receptor antagonist; Synergen/Amgen); anakinra (Kineret®/Amgen);TNF-bp/s-TNF (soluble TNF binding protein; see e.g., Evans et al.(Abstract No. 1540), Arthritis Rheum., 39(9)(supplement): S284 (1996));Kapadia et al., Amer. J. Physiol.—Heart and Circulatory Physiology, 268:H517-H525 (1995)); RP73401 (phosphodiesterase Type IV inhibitor; seee.g., Chikanza et al. (Abstract No. 1527), Arthritis Rheum., 39(9)(supplement): 5282 (1996)); MK-966 (COX-2 Inhibitor; see e.g., Erich etal. (Abstract Nos. 328 and 329), Arthritis Rheum., 39(9)(supplement):S81 (1996)); Iloprost (see e.g., Scholz, P. (Abstract No. 336),Arthritis Rheum., 39(9) (supplement): S82 (1996)); methotrexate;thalidomide (see e.g., Lee et al. (Abstract No. 1524), Arthritis Rheum.,39(9)(supplement): 5282 (1996)) and thalidomide-related drugs (e.g.,Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; seee.g., Finnegan et al. (Abstract No. 627), Arthritis Rheum.,39(9)(supplement): 5131 (1996)); Thoss et al., Inflamm. Res., 45:103-107 (1996)); tranexamic acid (inhibitor of plasminogen activation;see e.g., Ronday et al. (Abstract No. 1541), Arthritis Rheum.,39(9)(supplement): 5284 (1996)); T-614 (cytokine inhibitor; see e.g.,Hara et al. (Abstract No. 1526), Arthritis Rheum., 39(9)(supplement):5282 (1996)); prostaglandin E1 (see e.g., Moriuchi et al. (Abstract No.1528), Arthritis Rheum., 39(9)(supplement): 5282 (1996)); Tenidap(non-steroidal anti-inflammatory drug; see e.g., Guttadauria, M.(Abstract No. 1516), Arthritis Rheum., 39(9)(supplement): 5280 (1996));Naproxen (non-steroidal anti-inflammatory drug; see e.g., Fiebich etal., Neuro Report, 7: 1209-1213 (1996)); Meloxicam (non-steroidalanti-inflammatory drug); Ibuprofen (non-steroidal anti-inflammatorydrug); Piroxicam (non-steroidal anti-inflammatory drug); Diclofenac(non-steroidal anti-inflammatory drug); Indomethacin (non-steroidalanti-inflammatory drug); Sulfasalazine (see e.g., Farr et al. (AbstractNo. 1519), Arthritis Rheum., 39(9)(supplement): S281 (1996));Azathioprine (see e.g., Hickey et al. (Abstract No. 1521), ArthritisRheum., 39(9)(supplement): S281 (1996)); ICE inhibitor (inhibitor of theenzyme interleukin-1β converting enzyme); zap-70 and/or lck inhibitor(inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/orVEGF-R inhibitor (inhibitors of vascular endothelial cell growth factoror vascular endothelial cell growth factor receptor; inhibitors ofangiogenesis); corticosteroid anti-inflammatory drugs (e.g., SB203580);TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies;interleukin—11 (see e.g., Keith Jr. et al. (Abstract No. 1613),Arthritis Rheum., 39(9)(supplement): 5296 (1996)); interleukin-13 (seee.g., Bessis et al. (Abstract No. 1681), Arthritis Rheum.,39(9)(supplement): 5308 (1996)); interleukin-17 inhibitors (see e.g.,Lotz et al. (Abstract No. 559), Arthritis Rheum., 39(9)(supplement):5120 (1996)); gold; penicillamine; chloroquine; chlorambucil;hydroxychloroquine; cyclosporine; cyclophosphamide; total lymphoidirradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally-administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals,Inc.); ICAM-1 antisense phosphorothioate oligo-deoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; see e.g., DeLuca et al., Rheum.Dis. Clin. North Am., 21: 759-777 (1995)); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506);sirolimus (rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko etal., J. Med. Chem., 50(4), 641-662 (2007)); antivirals and immunemodulating agents.

In one embodiment, the binding protein or antigen-binding portionthereof, is administered in combination with one of the following agentsfor the treatment of rheumatoid arthritis (RA): small molecule inhibitorof KDR, small molecule inhibitor of Tie-2; methotrexate; prednisone;celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib;etanercept; infliximab; leflunomide; naproxen; valdecoxib;sulfasalazine; methylprednisolone; ibuprofen; meloxicam;methylprednisolone acetate; gold sodium thiomalate; aspirin;azathioprine; triamcinolone acetonide; propoxyphene napsylate/apap;folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium;oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenacsodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl;salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen;alendronate sodium; prednisolone; morphine sulfate; lidocainehydrochloride; indomethacin; glucosamine sulfate/chondroitin;cyclosporine; amitriptyline hcl; sulfadiazine; oxycodonehcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium;omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15;BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485;CDC-801; and mesopram.

Non-limiting examples of therapeutic agents for inflammatory boweldisease with which a binding protein of the invention can be combinedinclude the following: budenoside; epidermal growth factor;corticosteroids; cyclosporin, sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1β mAbs; anti-IL-6 mAbs;growth factors; elastase inhibitors; pyridinyl-imidazole compounds;antibodies to or antagonists of other human cytokines or growth factors,for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-17,IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the invention, orantigen binding portions thereof, can be combined with antibodies tocell surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,CD40, CD45, CD69, CD90 or their ligands. The antibodies of theinvention, or antigen binding portions thereof, may also be combinedwith agents, such as methotrexate, cyclosporin, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adenosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signaling byproinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK,p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TNFαconverting enzyme inhibitors, T-cell signaling inhibitors such as kinaseinhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors, sIL-1R1, sIL-1R11, sIL-6R) and anti-inflammatory cytokines(e.g., IL-4, IL-10, IL-11, IL-13 and TGFβ) and bcl-2 inhibitors.

Examples of therapeutic agents for Crohn's disease in which a bindingprotein can be combined

include the following: TNF antagonists, for example, anti-TNFantibodies, Adalimumab (PCT Publication No. WO 97/29131; HUMIRA®), CA2(REMICADE), CDP 571, TNFR-Ig constructs, (p75TNFRIgG (ENBREL®) andp55TNFRIgG (LENERCEPT™)) inhibitors and PDE4 inhibitors. Antibodies ofthe invention, or antigen binding portions thereof, can be combined withcorticosteroids, for example, budenoside and dexamethasone. Bindingproteins of the invention or antigen binding portions thereof, may alsobe combined with agents such as sulfasalazine, 5-aminosalicylic acid andolsalazine, and agents which interfere with synthesis or action ofproinflammatory cytokines such as IL-1, for example, IL-1 convertingenzyme inhibitors and IL-1ra. Antibodies of the invention or antigenbinding portion thereof may also be used with T cell signalinginhibitors, for example, tyrosine kinase inhibitors 6-mercaptopurines.Binding proteins of the invention, or antigen binding portions thereof,can be combined with IL-11. Binding proteins of the invention, orantigen binding portions thereof, can be combined with mesalamine,prednisone, azathioprine, mercaptopurine, infliximab, methylprednisolonesodium succinate, diphenoxylate/atrop sulfate, loperamide hydrochloride,methotrexate, omeprazole, folate, ciprofloxacin/dextrose-water,hydrocodone bitartrate/apap, tetracycline hydrochloride, fluocinonide,metronidazole, thimerosal/boric acid, cholestyramine/sucrose,ciprofloxacin hydrochloride, hyoscyamine sulfate, meperidinehydrochloride, midazolam hydrochloride, oxycodone hcl/acetaminophen,promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin, folic acid,levofloxacin, methylprednisolone, natalizumab and interferon-gamma.

Non-limiting examples of therapeutic agents for multiple sclerosis (MS)with which binding proteins of the invention can be combined include thefollowing: corticosteroids; prednisolone; methylprednisolone;azathioprine; cyclophosphamide; cyclosporine; methotrexate;4-aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen);interferon-β1b (BETASERON; Chiron/Berlex); interferon α-n3) (InterferonSciences/Fujimoto), interferon-α (Alfa Wassermann/J&J), interferonβ1A-1F (Serono/Inhale Therapeutics), Peginterferon α 2b(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; TevaPharmaceutical Industries, Inc.); hyperbaric oxygen; intravenousimmunoglobulin; clabribine; antibodies to or antagonists of other humancytokines or growth factors and their receptors, for example, TNF, LT,IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, and PDGF. Binding proteins of the invention can be combinedwith antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,CD19, CD₂O, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 ortheir ligands. Binding proteins of the invention, may also be combinedwith agents, such as methotrexate, cyclosporine, FK506, rapamycin,mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,corticosteroids such as prednisolone, phosphodiesterase inhibitors,adensosine agonists, antithrombotic agents, complement inhibitors,adrenergic agents, agents which interfere with signaling byproinflammatory cytokines such as TNFα or IL-1 (e.g., IRAK, NIK, IKK,p38 or MAP kinase inhibitors), IL-1β converting enzyme inhibitors, TACEinhibitors, T-cell signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNFreceptors, sIL-1R1, sIL-1R11, sIL-6R), anti-inflammatory cytokines(e.g., IL-4, IL-10, IL-13 and TGFβ) and bcl-2 inhibitors.

Examples of therapeutic agents for multiple sclerosis with which bindingproteins of the invention can be combined include interferon-β, forexample, IFNβ1a and IFNβ1b; copaxone; corticosteroids; caspaseinhibitors, for example inhibitors of caspase-1; IL-1 inhibitors; TNFinhibitors; and antibodies to CD40 ligand and CD80.

The binding proteins of the invention, may also be combined with agents,such as alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone,xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab,sinnabidol, a-immunokine NNSO3, ABR-215062, AnergiX.MS, chemokinereceptor antagonists, BBR-2778, calagualine, CPI-1189, LEM (liposomeencapsulated mitoxantrone), THC.CBD (cannabinoid agonist) MBP-8298,mesopram (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody,neurovax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, talampanel,teriflunomide, TGF-beta2, tiplimotide, VLA-4 antagonists (for example,TR-14035, VLA4 Ultrahaler, Antegran-ELAN/Biogen), interferon gammaantagonists, IL-4 agonists.

Non-limiting examples of therapeutic agents for angina with whichbinding proteins of the invention can be combined include the following:aspirin, nitroglycerin, isosorbide mononitrate, metoprolol succinate,atenolol, metoprolol tartrate, amlodipine besylate, diltiazemhydrochloride, isosorbide dinitrate, clopidogrel bisulfate, nifedipine,atorvastatin calcium, potassium chloride, furosemide, simvastatin,verapamil hcl, digoxin, propranolol hydrochloride, carvedilol,lisinopril, spironolactone, hydrochlorothiazide, enalapril maleate,nadolol, ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalolhydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium,lisinopril/hydrochlorothiazide, felodipine, captopril, bisoprololfumarate.

Non-limiting examples of therapeutic agents for ankylosing spondylitiswith which binding proteins of the invention can be combined include thefollowing: ibuprofen, diclofenac and misoprostol, naproxen, meloxicam,indomethacin, diclofenac, celecoxib, rofecoxib, sulfasalazine,methotrexate, azathioprine, minocyclin, prednisone, etanercept,infliximab.

Non-limiting examples of therapeutic agents for asthma with whichbinding proteins of the invention can be combined include the following:albuterol, salmeterol/fluticasone, montelukast sodium, fluticasonepropionate, budesonide, prednisone, salmeterol xinafoate, levalbuterolhcl, albuterol sulfate/ipratropium, prednisolone sodium phosphate,triamcinolone acetonide, beclomethasone dipropionate, ipratropiumbromide, azithromycin, pirbuterol acetate, prednisolone, theophyllineanhydrous, methylprednisolone sodium succinate, clarithromycin,zafirlukast, formoterol fumarate, influenza virus vaccine,methylprednisolone, amoxicillin trihydrate, flunisolide, allergyinjection, cromolyn sodium, fexofenadine hydrochloride,flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhalerassist device, guaifenesin, dexamethasone sodium phosphate, moxifloxacinhcl, doxycycline hyclate, guaifenesin/d-methorphan,p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine hydrochloride,mometasone furoate, salmeterol xinafoate, benzonatate, cephalexin,pe/hydrocodone/chlorphenir, cetirizine hcl/pseudoephed,phenylephrine/cod/promethazine, codeine/promethazine, cefprozil,dexamethasone, guaifenesin/pseudoephedrine,chlorpheniramine/hydrocodone, nedocromil sodium, terbutaline sulfate,epinephrine, methylprednisolone, metaproterenol sulfate.

Non-limiting examples of therapeutic agents for COPD with which bindingproteins of the invention can be combined include the following:albuterol sulfate/ipratropium, ipratropium bromide,salmeterol/fluticasone, albuterol, salmeterol xinafoate, fluticasonepropionate, prednisone, theophylline anhydrous, methylprednisolonesodium succinate, montelukast sodium, budesonide, formoterol fumarate,triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin,beclomethasone dipropionate, levalbuterol hcl, flunisolide, ceftriaxonesodium, amoxicillin trihydrate, gatifloxacin, zafirlukast,amoxicillin/clavulanate, flunisolide/menthol,chlorpheniramine/hydrocodone, metaproterenol sulfate,methylprednisolone, mometasone furoate, p-ephedrine/cod/chlorphenir,pirbuterol acetate, p-ephedrine/loratadine, terbutaline sulfate,tiotropium bromide, (R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.

Non-limiting examples of therapeutic agents for HCV with which bindingproteins of the invention can be combined include the following:Interferon-alpha-2a, Interferon-alpha-2b, Interferon-alpha con1,Interferon-alpha-n1, Pegylated interferon-alpha-2a, Pegylatedinterferon-alpha-2b, ribavirin, Peginterferon alfa-2b+ribavirin,Ursodeoxycholic Acid, Glycyrrhizic Acid, Thymalfasin, Maxamine, VX-497and any compounds that are used to treat HCV through intervention withthe following targets: HCV polymerase, HCV protease, HCV helicase, HCVIRES (internal ribosome entry site).

Non-limiting examples of therapeutic agents for idiopathic pulmonaryfibrosis with which binding proteins of the invention can be combinedinclude the following: prednisone, azathioprine, albuterol, colchicine,albuterol sulfate, digoxin, gamma interferon, methylprednisolone sodsucc, lorazepam, furosemide, lisinopril, nitroglycerin, spironolactone,cyclophosphamide, ipratropium bromide, actinomycin d, alteplase,fluticasone propionate, levofloxacin, metaproterenol sulfate, morphinesulfate, oxycodone hcl, potassium chloride, triamcinolone acetonide,tacrolimus anhydrous, calcium, interferon-alpha, methotrexate,mycophenolate mofetil, Interferon-gamma-1β.

Non-limiting examples of therapeutic agents for myocardial infarctionwith which binding proteins of the invention can be combined include thefollowing: aspirin, nitroglycerin, metoprolol tartrate, enoxaparinsodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol,morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril,isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril,tenecteplase, enalapril maleate, torsemide, retavase, losartanpotassium, quinapril hcl/mag carb, bumetanide, alteplase, enalaprilat,amiodarone hydrochloride, tirofiban hcl m-hydrate, diltiazemhydrochloride, captopril, irbesartan, valsartan, propranololhydrochloride, fosinopril sodium, lidocaine hydrochloride, eptifibatide,cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone,interferon, sotalol hydrochloride, potassium chloride, docusate sodium,dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin calcium,midazolam hydrochloride, meperidine hydrochloride, isosorbide dinitrate,epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,ezetimibe/simvastatin, avasimibe, cariporide.

Non-limiting examples of therapeutic agents for psoriasis with whichbinding proteins of the invention can be combined include the following:small molecule inhibitor of KDR, small molecule inhibitor of Tie-2,calcipotriene, clobetasol propionate, triamcinolone acetonide,halobetasol propionate, tazarotene, methotrexate, fluocinonide,betamethasone diprop augmented, fluocinolone acetonide, acitretin, tarshampoo, betamethasone valerate, mometasone furoate, ketoconazole,pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea,betamethasone, clobetasol propionate/emoll, fluticasone propionate,azithromycin, hydrocortisone, moisturizing formula, folic acid,desonide, pimecrolimus, coal tar, diflorasone diacetate, etanerceptfolate, lactic acid, methoxsalen, hc/bismuth subgal/znox/resor,methylprednisolone acetate, prednisone, sunscreen, halcinonide,salicylic acid, anthralin, clocortolone pivalate, coal extract, coaltar/salicylic acid, coal tar/salicylic acid/sulfur, desoximetasone,diazepam, emollient, fluocinonide/emollient, mineral oil/castor oil/nalact, mineral oil/peanut oil, petroleum/isopropyl myristate, psoralen,salicylic acid, soap/tribromsalan, thimerosal/boric acid, celecoxib,infliximab, cyclosporine, alefacept, efalizumab, tacrolimus,pimecrolimus, PUVA, UVB, sulfasalazine.

Non-limiting examples of therapeutic agents for psoriatic arthritis withwhich binding proteins of the invention can be combined include thefollowing: methotrexate, etanercept, rofecoxib, celecoxib, folic acid,sulfasalazine, naproxen, leflunomide, methylprednisolone acetate,indomethacin, hydroxychloroquine sulfate, prednisone, sulindac,betamethasone diprop augmented, infliximab, methotrexate, folate,triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam,diclofenac sodium, ketoprofen, meloxicam, methylprednisolone,nabumetone, tolmetin sodium, calcipotriene, cyclosporine, diclofenacsodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodiumthiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate sodium,sulfadiazine, thioguanine, valdecoxib, alefacept, efalizumab and bcl-2inhibitors.

Non-limiting examples of therapeutic agents for restenosis with whichbinding proteins of the invention can be combined include the following:sirolimus, paclitaxel, everolimus, tacrolimus, Zotarolimus,acetaminophen.

Non-limiting examples of therapeutic agents for sciatica with whichbinding proteins of the invention can be combined include the following:hydrocodone bitartrate/apap, rofecoxib, cyclobenzaprine hcl,methylprednisolone, naproxen, ibuprofen, oxycodone hcl/acetaminophen,celecoxib, valdecoxib, methylprednisolone acetate, prednisone, codeinephosphate/apap, tramadol hcl/acetaminophen, metaxalone, meloxicam,methocarbamol, lidocaine hydrochloride, diclofenac sodium, gabapentin,dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin,acetaminophen, diazepam, nabumetone, oxycodone hcl, tizanidine hcl,diclofenac sodium/misoprostol, propoxyphene napsylate/apap,asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeinephos/asa, morphine sulfate, multivitamins, naproxen sodium, orphenadrinecitrate, temazepam.

Examples of therapeutic agents for SLE (lupus) with which bindingproteins of the invention can be combined include the following: NSAIDS,for example, diclofenac, naproxen, ibuprofen, piroxicam, indomethacin;COX2 inhibitors, for example, Celecoxib, rofecoxib, valdecoxib;anti-malarials, for example, hydroxychloroquine; Steroids, for example,prednisone, prednisolone, budenoside, dexamethasone; Cytotoxics, forexample, azathioprine, cyclophosphamide, mycophenolate mofetil,methotrexate; inhibitors of PDE4 or purine synthesis inhibitor, forexample Cellcept. Binding proteins of the invention, may also becombined with agents such as sulfasalazine, 5-aminosalicylic acid,olsalazine, Imuran and agents which interfere with synthesis, productionor action of proinflammatory cytokines such as IL-1, for example,caspase inhibitors like IL-1β converting enzyme inhibitors and IL-1ra.Binding proteins of the invention may also be used with T cell signalinginhibitors, for example, tyrosine kinase inhibitors; or molecules thattarget T cell activation molecules, for example, CTLA-4-IgG or anti-B7family antibodies, anti-PD-1 family antibodies. Binding proteins of theinvention, can be combined with IL-11 or anti-cytokine antibodies, forexample, fonotolizumab (anti-IFNg antibody), or anti-receptor receptorantibodies, for example, anti-IL-6 receptor antibody and antibodies toB-cell surface molecules. Antibodies of the invention or antigen bindingportion thereof may also be used with LJP 394 (abetimus), agents thatdeplete or inactivate B-cells, for example, Rituximab (anti-CD₂Oantibody), lymphostat-B (anti-BlyS antibody), TNF antagonists, forexample, anti-TNF antibodies, Adalimumab (PCT Publication No. WO97/29131; HUMIRA®), CA2 (REMICADE®), CDP 571, TNFR-Ig constructs,(p75TNFRIgG (ENBREL®) and p55TNFRIgG (LENERCEPT®)) and bcl-2 inhibitors,because bcl-2 overexpression in transgenic mice has been demonstrated tocause a lupus like phenotype (see Marquina et al., J. Immunol., 172(11):7177-7185 (2004)), therefore inhibition is expected to have therapeuticeffects.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodyportion to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody, or antibody portion, are outweighedby the therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

Diagnostics

The disclosure herein also provides diagnostic applications. This isfurther elucidated below. Antibodies that bind IL-1β of the inventionmay be employed in any of a variety of formats to detect IL-1β in vivo,in vitro, or ex vivo (i.e., in cells or tissues that have been obtainedfrom a living individual, subjected to a procedure, then returned to theindividual). DVD-Igs of the invention offer the further advantage ofbeing capable of binding to an epitope of IL-1β as well as otherantigens or epitopes in various diagnostic and detection assay formats.

I. Method of Assay

The present disclosure also provides a method for determining thepresence, amount or concentration of an IL-113, or a fragment thereof,(“analyte”) in a test sample using at least one anti-IL-1β bindingprotein or antigen binding portion thereof, including a DVD-Ig, asdescribed herein. Any suitable assay as is known in the art can be usedin the method. Examples include, but are not limited to, immunoassay,such as sandwich immunoassay (e.g., monoclonal, polyclonal and/or DVD-Igsandwich immunoassays or any variation thereof (e.g., monoclonal/DVD-Ig,DVD-Ig/polyclonal, etc.), including radioisotope detection(radioimmunoassay (RIA)) and enzyme detection (enzyme immunoassay (EIA)or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISAassays, R&D Systems, Minneapolis, Minn.)), competitive inhibitionimmunoassay (e.g., forward and reverse), fluorescence polarizationimmunoassay (FPIA), enzyme multiplied immunoassay technique (EMIT),bioluminescence resonance energy transfer (BRET), and homogeneouschemiluminescent assay, etc. In a SELDI-based immunoassay, a capturereagent that specifically binds an analyte (or a fragment thereof) ofinterest is attached to the surface of a mass spectrometry probe, suchas a pre-activated protein chip array. The analyte (or a fragmentthereof) is then specifically captured on the biochip, and the capturedanalyte (or a fragment thereof) is detected by mass spectrometry.Alternatively, the analyte (or a fragment thereof) can be eluted fromthe capture reagent and detected by traditional MALDI (matrix-assistedlaser desorption/ionization) or by SELDI. A chemiluminescentmicroparticle immunoassay, in particular one employing the ARCHITECT®automated analyzer (Abbott Laboratories, Abbott Park, Ill.), is anexample of an exemplary immunoassay.

Methods well-known in the art for collecting, handling and processingurine, blood, serum and plasma, and other body fluids, are used in thepractice of the present disclosure, for instance, when anti-IL-1βbinding protein as described herein is employed as an immunodiagnosticreagent and/or in an analyte immunoassay kit. The test sample cancomprise further moieties in addition to the analyte of interest, suchas antibodies, antigens, haptens, hormones, drugs, enzymes, receptors,proteins, peptides, polypeptides, oligonucleotides and/orpolynucleotides. For example, the sample can be a whole blood sampleobtained from a subject. It can be necessary or desired that a testsample, particularly whole blood, be treated prior to immunoassay asdescribed herein, e.g., with a pretreatment reagent. Even in cases wherepretreatment is not necessary (e.g., most urine samples), pretreatmentoptionally can be done (e.g., as part of a regimen on a commercialplatform).

The pretreatment reagent can be any reagent appropriate for use with theimmunoassay and kits of the invention. The pretreatment optionallycomprises: (a) one or more solvents (e.g., methanol and ethylene glycol)and optionally, salt, (b) one or more solvents and salt, and optionally,detergent, (c) detergent, or (d) detergent and salt. Pretreatmentreagents are known in the art, and such pretreatment can be employed,e.g., as used for assays on Abbott TDx, AxSYM®, and ARCHITECT® analyzers(Abbott Laboratories, Abbott Park, Ill.), as described in the literature(see, e.g., Yatscoff et al., “Abbott TDx Monoclonal Antibody AssayEvaluated for Measuring Cyclosporine in Whole Blood,” Clin. Chem., 36:1969-1973 (1990); and Wallemacq et al., “Evaluation of the New AxSYMCyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and EMITCyclosporine Assays,” Clin. Chem., 45: 432-435 (1999)), and/or ascommercially available. Additionally, pretreatment can be done asdescribed in Abbott's U.S. Pat. No. 5,135,875; European Publication No.EP 0 471 293; PCT Publication No. WO 2008/082984; and US Publication No.2008/0020401 (incorporated by reference in its entirety for itsteachings regarding pretreatment). The pretreatment reagent can be aheterogeneous agent or a homogeneous agent.

With use of a heterogeneous pretreatment reagent, the pretreatmentreagent precipitates analyte binding protein (e.g., protein that canbind to an analyte or a fragment thereof) present in the sample. Such apretreatment step comprises removing any analyte binding protein byseparating from the precipitated analyte binding protein the supernatantof the mixture formed by addition of the pretreatment agent to sample.In such an assay, the supernatant of the mixture absent any bindingprotein is used in the assay, proceeding directly to the antibodycapture step.

With use of a homogeneous pretreatment reagent there is no suchseparation step. The entire mixture of test sample and pretreatmentreagent are contacted with a labeled specific binding partner foranalyte (or a fragment thereof), such as a labeled anti-analyte antibody(or an antigenically reactive fragment thereof). The pretreatmentreagent employed for such an assay typically is diluted in thepretreated test sample mixture, either before or during capture by thefirst specific binding partner. Despite such dilution, a certain amountof the pretreatment reagent is still present (or remains) in the testsample mixture during capture. According to the invention, an exemplarylabeled specific binding partner can be a DVD-Ig (or a fragment, avariant, or a fragment of a variant thereof).

In a heterogeneous format, after the test sample is obtained from asubject, a first mixture is prepared. The mixture contains the testsample being assessed for an analyte (or a fragment thereof) and a firstspecific binding partner, wherein the first specific binding partner andany analyte contained in the test sample form a first specific bindingpartner-analyte complex. Preferably, the first specific binding partneris an anti-analyte antibody or a fragment thereof. The first specificbinding partner can be a DVD-Ig (or a fragment, a variant, or a fragmentof a variant thereof) as described herein. The order in which the testsample and the first specific binding partner are added to form themixture is not critical. Preferably, the first specific binding partneris immobilized on a solid phase. The solid phase used in the immunoassay(for the first specific binding partner and, optionally, the secondspecific binding partner) can be any solid phase known in the art, suchas, but not limited to, a magnetic particle, a bead, a test tube, amicrotiter plate, a cuvette, a membrane, a scaffolding molecule, a film,a filter paper, a disc and a chip.

After the mixture containing the first specific binding partner-analytecomplex is formed, any unbound analyte is removed from the complex usingany technique known in the art. For example, the unbound analyte can beremoved by washing. Desirably, however, the first specific bindingpartner is present in excess of any analyte present in the test sample,such that all analyte that is present in the test sample is bound by thefirst specific binding partner.

After any unbound analyte is removed, a second specific binding partneris added to the mixture to form a first specific bindingpartner-analyte-second specific binding partner complex. The secondspecific binding partner is preferably an anti-analyte antibody thatbinds to an epitope on analyte that differs from the epitope on analytebound by the first specific binding partner. Moreover, also preferably,the second specific binding partner is labeled with or contains adetectable label as described above. The second specific binding partnercan be a DVD-Ig (or a fragment, a variant, or a fragment of a variantthereof) as described herein.

Any suitable detectable label as is known in the art can be used. Forexample, the detectable label can be a radioactive label (such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, and ³³P), an enzymatic label (such as horseradishperoxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, andthe like), a chemiluminescent label (such as acridinium esters,thioesters, or sulfonamides; luminol, isoluminol, phenanthridiniumesters, and the like), a fluorescent label (such as fluorescein (e.g.,5-fluorescein, 6-carboxyfluorescein, 3′6 -carboxyfluorescein,5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)),rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zincsulfide-capped cadmium selenide), a thermometric label, or animmuno-polymerase chain reaction label. An introduction to labels,labeling procedures and detection of labels is found in Polak and VanNoorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag,N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes andResearch Chemicals (1996), which is a combined handbook and cataloguepublished by Molecular Probes, Inc., Eugene, Oreg. A fluorescent labelcan be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896; 5,573,904;5,496,925; 5,359,093, and 5,352,803). An acridinium compound can be usedas a detectable label in a homogeneous or heterogeneous chemiluminescentassay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett., 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett., 14:2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Lett., 14:3917-3921 (2004); and Adamczyk et al., Org. Lett., 5: 3779-3782 (2003)).

An exemplary acridinium compound is an acridinium-9-carboxamide. Methodsfor preparing acridinium 9-carboxamides are described in Mattingly, J.Biolumin. Chemilumin., 6: 107-114 (1991); Adamczyk et al., J. Org.Chem., 63: 5636-5639 (1998); Adamczyk et al., Tetrahedron, 55:10899-10914 (1999); Adamczyk et al., Org. Lett., 1: 779-781 (1999);Adamczyk et al., Bioconjugate Chem., 11: 714-724 (2000); Adamczyk andMattingly, In Luminescence Biotechnology: Instruments and Applications;(Dyke, K. V., ed.) (CRC Press: Boca Raton, 2002) pp. 77-105; Adamczyk etal., Org. Lett., 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646,5,543,524 and 5,783,699. Another preferred acridinium compound is anacridinium-9-carboxylate aryl ester. An example of anacridinium-9-carboxylate aryl ester is10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available fromCayman Chemical, Ann Arbor, Mich.). Methods for preparing acridinium9-carboxylate aryl esters are described in McCapra et al., Photochem.Photobiol., 4: 1111-21 (1965); Razavi et al., Luminescence, 15: 245-249(2000); Razavi et al., Luminescence, 15: 239-244 (2000); and U.S. Pat.No. 5,241,070. Further details regarding acridinium-9-carboxylate arylester and its use are set forth in US Publication No. 2008/0248493.

Chemiluminescent assays (e.g., using acridinium as described above orother chemiluminescent agents) can be performed in accordance with themethods described in Adamczyk et al., Anal. Chim. Acta, 579(1): 61-67(2006). While any suitable assay format can be used, a microplatechemiluminometer (Mithras LB-940, Berthold Technologies USA., LLC, OakRidge, Tenn.) enables the assay of multiple samples of small volumesrapidly.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for chemiluminescent assay is notcritical. If the first specific binding partner is detectably labeledwith a chemiluminescent agent such as an acridinium compound, detectablylabeled first specific binding partner-analyte complexes form.Alternatively, if a second specific binding partner is used and thesecond specific binding partner is detectably labeled with achemiluminescent agent such as an acridinium compound, detectablylabeled first specific binding partner-analyte-second specific bindingpartner complexes form. Any unbound specific binding partner, whetherlabeled or unlabeled, can be removed from the mixture using anytechnique known in the art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided orsupplied to the mixture (e.g., the source of the hydrogen peroxide beingone or more buffers or other solutions that are known to containhydrogen peroxide) before, simultaneously with, or after the addition ofan above-described acridinium compound. Hydrogen peroxide can begenerated in situ in a number of ways such as would be apparent to oneskilled in the art.

Upon the simultaneous or subsequent addition of at least one basicsolution to the sample, a detectable signal, namely, a chemiluminescentsignal, indicative of the presence of analyte is generated. The basicsolution contains at least one base and has a pH greater than or equalto 10, preferably, greater than or equal to 12. Examples of basicsolutions include, but are not limited to, sodium hydroxide, potassiumhydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide,sodium carbonate, sodium bicarbonate, calcium hydroxide, calciumcarbonate, and calcium bicarbonate. The amount of basic solution addedto the sample depends on the concentration of the basic solution. Basedon the concentration of the basic solution used, one skilled in the artcan easily determine the amount of basic solution to add to the sample.

The chemiluminescent signal that is generated can be detected usingroutine techniques known to those skilled in the art. Based on theintensity of the signal generated, the amount of analyte in the samplecan be quantified. Specifically, the amount of analyte in the sample isproportional to the intensity of the signal generated. The amount ofanalyte present can be quantified by comparing the amount of lightgenerated to a standard curve for analyte or by comparison to areference standard. The standard curve can be generated using serialdilutions or solutions of known concentrations of analyte by massspectroscopy, gravimetric methods, and other techniques known in theart. While the above is described with emphasis on use of an acridiniumcompound as the chemiluminescent agent, one of ordinary skill in the artcan readily adapt this description for use of other chemiluminescentagents.

Analyte immunoassays generally can be conducted using any format knownin the art, such as, but not limited to, a sandwich format.Specifically, in one immunoassay format, at least two antibodies areemployed to separate and quantify analyte, such as human analyte, or afragment thereof in a sample. More specifically, the at least twoantibodies bind to different epitopes on an analyte (or a fragmentthereof) forming an immune complex, which is referred to as a“sandwich.” Generally, in the immunoassays one or more antibodies can beused to capture the analyte (or a fragment thereof) in the test sample(these antibodies are frequently referred to as a “capture” antibody or“capture” antibodies) and one or more antibodies can be used to bind adetectable (namely, quantifiable) label to the sandwich (theseantibodies are frequently referred to as the “detection antibody,” the“detection antibodies,” the “conjugate,” or the “conjugates”). Thus, inthe context of a sandwich immunoassay format, a DVD-Ig (or a fragment, avariant, or a fragment of a variant thereof) as described herein can beused as a capture antibody, a detection antibody, or both. For example,one DVD-Ig having a domain that can bind a first epitope on an analyte(or a fragment thereof) can be used as a capture antibody and/or anotherDVD-Ig having a domain that can bind a second epitope on an analyte (ora fragment thereof) can be used as a detection antibody. In this regard,a DVD-Ig having a first domain that can bind a first epitope on ananalyte (or a fragment thereof) and a second domain that can bind asecond epitope on an analyte (or a fragment thereof) can be used as acapture antibody and/or a detection antibody. Alternatively, one DVD-Ighaving a first domain that can bind an epitope on a first analyte (or afragment thereof) and a second domain that can bind an epitope on asecond analyte (or a fragment thereof) can be used as a capture antibodyand/or a detection antibody to detect, and optionally quantify, two ormore analytes. In the event that an analyte can be present in a samplein more than one form, such as a monomeric form and a dimeric/multimericform, which can be homomeric or heteromeric, one DVD-Ig having a domainthat can bind an epitope that is only exposed on the monomeric form andanother DVD-Ig having a domain that can bind an epitope on a differentpart of a dimeric/multimeric form can be used as capture antibodiesand/or detection antibodies, thereby enabling the detection, andoptional quantification, of different forms of a given analyte.Furthermore, employing DVD-Igs with differential affinities within asingle DVD-Ig and/or between DVD-Igs can provide an avidity advantage.In the context of immunoassays as described herein, it generally may behelpful or desired to incorporate one or more linkers within thestructure of a DVD-Ig. When present, optimally the linker should be ofsufficient length and structural flexibility to enable binding of anepitope by the inner domains as well as binding of another epitope bythe outer domains. In this regard, if a DVD-Ig can bind two differentanalytes and one analyte is larger than the other, desirably the largeranalyte is bound by the outer domains.

Generally speaking, a sample being tested for (for example, suspected ofcontaining) an IL-1β protein (or a fragment thereof) can be contactedwith at least one capture antibody (or antibodies) and at least onedetection antibody (which can be a second detection antibody or a thirddetection antibody or even a successively numbered antibody, e.g., aswhere the capture and/or detection antibody comprise multipleantibodies) either simultaneously or sequentially and in any order. Forexample, the test sample can be first contacted with at least onecapture antibody and then (sequentially) with at least one detectionantibody. Alternatively, the test sample can be first contacted with atleast one detection antibody and then (sequentially) with at least onecapture antibody. In yet another alternative, the test sample can becontacted simultaneously with a capture antibody and a detectionantibody.

In the sandwich assay format, a sample suspected of containing IL-1β (ora fragment thereof) is first brought into contact with at least onefirst capture binding protein (e.g., IL-1β antibody) under conditionsthat allow the formation of a first binding protein/IL-1β complex. Ifmore than one capture binding protein is used, a first capture bindingprotein/IL-1β complex comprising two or more capture binding proteinsforms. In a sandwich assay, the binding proteins, i.e., preferably, theat least one capture binding protein, are used in molar excess amountsof the maximum amount of IL-1β analyte (or a fragment thereof) expectedin the test sample. For example, from about 5 μg to about 1 mg ofantibody per mL of buffer (e.g., microparticle coating buffer) can beused.

Competitive inhibition immunoassays, which are often used to measuresmall analytes because binding by only one antibody is required,comprise sequential and classic formats. In a sequential competitiveinhibition immunoassay a capture binding protein to IL-1β is coated ontoa well of a microtiter plate or other solid support. When the samplecontaining the IL-1β is added to the well, the IL-1β binds to thecapture binding protein. After washing, a known amount of labeled (e.g.,biotin or horseradish peroxidase (HRP)) IL-1β is added to the well. Asubstrate for an enzymatic label is necessary to generate a signal. Anexample of a suitable substrate for HRP is3,3′,5,5′-tetramethylbenzidine (TMB). After washing, the signalgenerated by the labeled analyte is measured and is inverselyproportional to the amount of IL-1β in the sample. In a classiccompetitive inhibition immunoassay, a binding protein to IL-1β is coatedonto a solid support (e.g., a well of a microtiter plate). However,unlike the sequential competitive inhibition immunoassay, the sample andthe labeled IL-1β are added to the well at the same time. Any IL-1β inthe sample competes with labeled IL-1β for binding to the capturebinding protein. After washing, the signal generated by the labeledIL-1β is measured and is inversely proportional to the amount of IL-1βin the sample.

Optionally, prior to contacting the test sample with the at least onecapture binding protein (for example, the first capture antibody), theat least one capture binding protein can be bound to a solid support,which facilitates the separation of the first binding protein/IL-1β (ora fragment thereof) complex from the test sample. The substrate to whichthe capture binding protein is bound can be any suitable solid supportor solid phase that facilitates separation of the captureantibody-analyte complex from the sample.

Examples include a well of a plate, such as a microtiter plate, a testtube, a porous gel (e.g., silica gel, agarose, dextran, or gelatin), apolymeric film (e.g., polyacrylamide), beads (e.g., polystyrene beads ormagnetic beads), a strip of a filter/membrane (e.g., nitrocellulose ornylon), microparticles (e.g., latex particles, magnetizablemicroparticles (e.g., microparticles having ferric oxide or chromiumoxide cores and homo- or hetero-polymeric coats and radii of about 1-10microns). The substrate can comprise a suitable porous material with asuitable surface affinity to bind antigens and sufficient porosity toallow access by detection antibodies. A microporous material isgenerally preferred, although a gelatinous material in a hydrated statecan be used. Such porous substrates are preferably in the form of sheetshaving a thickness of about 0.01 to about 0.5 mm, preferably about 0.1mm. While the pore size may vary quite a bit, preferably the pore sizeis from about 0.025 to about 15 microns, more preferably from about 0.15to about 15 microns. The surface of such substrates can be activated bychemical processes that cause covalent linkage of an antibody to thesubstrate. Irreversible binding, generally by adsorption throughhydrophobic forces, of the antigen or the antibody to the substrateresults; alternatively, a chemical coupling agent or other means can beused to bind covalently the antibody to the substrate, provided thatsuch binding does not interfere with the ability of the antibody to bindto analyte. Alternatively, the antibody can be bound withmicroparticles, which have been previously coated with streptavidin(e.g., DYNAL® Magnetic Beads, Invitrogen, Carlsbad, Calif.) or biotin(e.g., using Power-Bind™-SA-MP streptavidin-coated microparticles(Seradyn, Indianapolis, Ind.)) or anti-species-specific monoclonalantibodies. If necessary, the substrate can be derivatized to allowreactivity with various functional groups on the antibody. Suchderivatization requires the use of certain coupling agents, examples ofwhich include, but are not limited to, maleic anhydride,N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. If desired, one or more capture reagents, such asantibodies (or fragments thereof), each of which is specific foranalyte(s) can be attached to solid phases in different physical oraddressable locations (e.g., such as in a biochip configuration (see,e.g., U.S. Pat. No. 6,225,047; PCT Publication No. WO 99/51773; U.S.Pat. No. 6,329,209; PCT Publication No. WO 00/56934; and U.S. Pat. No.5,242,828). If the capture reagent is attached to a mass spectrometryprobe as the solid support, the amount of analyte bound to the probe canbe detected by laser desorption ionization mass spectrometry.Alternatively, a single column can be packed with different beads, whichare derivatized with the one or more capture reagents, thereby capturingthe analyte in a single place (see, antibody-derivatized, bead-basedtechnologies, e.g., the xMAP technology of Luminex (Austin, Tex.)).

After the test sample being assayed for analyte (or a fragment thereof)is brought into contact with the at least one capture antibody (forexample, the first capture antibody), the mixture is incubated in orderto allow for the formation of a first antibody (or multipleantibody)-analyte (or a fragment thereof) complex. The incubation can becarried out at a pH of from about 4.5 to about 10.0, at a temperature offrom about 2° C. to about 45° C., and for a period from at least aboutone (1) minute to about eighteen (18) hours, preferably from about 1 toabout 24 minutes, most preferably for about 4 to about 18 minutes. Theimmunoassay described herein can be conducted in one step (meaning thetest sample, at least one capture antibody and at least one detectionantibody are all added sequentially or simultaneously to a reactionvessel) or in more than one step, such as two steps, three steps, etc.

After formation of the (first or multiple) capture antibody/analyte (ora fragment thereof) complex, the complex is then contacted with at leastone detection antibody under conditions which allow for the formation ofa (first or multiple) capture antibody/analyte (or a fragmentthereof)/second detection antibody complex). While captioned for clarityas the “second” antibody (e.g., second detection antibody), in fact,where multiple antibodies are used for capture and/or detection, the atleast one detection antibody can be the second, third, fourth, etc.antibodies used in the immunoassay. If the capture antibody/analyte (ora fragment thereof) complex is contacted with more than one detectionantibody, then a (first or multiple) capture antibody/analyte (or afragment thereof)/(multiple) detection antibody complex is formed. Aswith the capture antibody (e.g., the first capture antibody), when theat least one (e.g., second and any subsequent) detection antibody isbrought into contact with the capture antibody/analyte (or a fragmentthereof) complex, a period of incubation under conditions similar tothose described above is required for the formation of the (first ormultiple) capture antibody/analyte (or a fragment thereof)/(second ormultiple) detection antibody complex. Preferably, at least one detectionantibody contains a detectable label. The detectable label can be boundto the at least one detection antibody (e.g., the second detectionantibody) prior to, simultaneously with, or after the formation of the(first or multiple) capture antibody/analyte (or a fragmentthereof)/(second or multiple) detection antibody complex. Any detectablelabel known in the art can be used (see discussion above, including ofthe Polak and Van Noorden (1997) and Haugland (1996) references).

The detectable label can be bound to the antibodies either directly orthrough a coupling agent. An example of a coupling agent that can beused is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,hydrochloride), which is commercially available from Sigma-Aldrich, St.Louis, Mo. Other coupling agents that can be used are known in the art.Methods for binding a detectable label to an antibody are known in theart. Additionally, many detectable labels can be purchased orsynthesized that already contain end groups that facilitate the couplingof the detectable label to the antibody, such as CPSP-Acridinium Ester(i.e., 9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridiniumcarboxamide) or SPSP-Acridinium Ester (i.e.,N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).

The (first or multiple) capture antibody/analyte/(second or multiple)detection antibody complex can be, but does not have to be, separatedfrom the remainder of the test sample prior to quantification of thelabel. For example, if the at least one capture antibody (e.g., thefirst capture antibody) is bound to a solid support, such as a well or abead, separation can be accomplished by removing the fluid (of the testsample) from contact with the solid support. Alternatively, if the atleast first capture antibody is bound to a solid support, it can besimultaneously contacted with the analyte-containing sample and the atleast one second detection antibody to form a first (multiple)antibody/analyte/second (multiple) antibody complex, followed by removalof the fluid (test sample) from contact with the solid support. If theat least one first capture antibody is not bound to a solid support,then the (first or multiple) capture antibody/analyte/(second ormultiple) detection antibody complex does not have to be removed fromthe test sample for quantification of the amount of the label.

After formation of the labeled capture antibody/analyte/detectionantibody complex (e.g., the first capture antibody/analyte/seconddetection antibody complex), the amount of label in the complex isquantified using techniques known in the art. For example, if anenzymatic label is used, the labeled complex is reacted with a substratefor the label that gives a quantifiable reaction such as the developmentof color. If the label is a radioactive label, the label is quantifiedusing appropriate means, such as a scintillation counter. If the labelis a fluorescent label, the label is quantified by stimulating the labelwith a light of one color (which is known as the “excitationwavelength”) and detecting another color (which is known as the“emission wavelength”) that is emitted by the label in response to thestimulation. If the label is a chemiluminescent label, the label isquantified by detecting the light emitted either visually or by usingluminometers, x-ray film, high speed photographic film, a CCD camera,etc. Once the amount of the label in the complex has been quantified,the concentration of analyte or a fragment thereof in the test sample isdetermined by appropriate means, such as by use of a standard curve thathas been generated using serial dilutions of analyte or a fragmentthereof of known concentration. Other than using serial dilutions ofanalyte or a fragment thereof, the standard curve can be generatedgravimetrically, by mass spectroscopy and by other techniques known inthe art.

In a chemiluminescent microparticle assay employing the ARCHITECT®analyzer, the conjugate diluent pH should be about 6.0+/−0.2, themicroparticle coating buffer should be maintained at about roomtemperature (i.e., at from about 17° C. to about 27° C.), themicroparticle coating buffer pH should be about 6.5+/−0.2, and themicroparticle diluent pH should be about 7.8+/−0.2. Solids preferablyare less than about 0.2%, such as less than about 0.15%, less than about0.14%, less than about 0.13%, less than about 0.12%, or less than about0.11%, such as about 0.10%.

FPIAs are based on competitive binding immunoassay principles. Afluorescently labeled compound, when excited by a linearly polarizedlight, will emit fluorescence having a degree of polarization inverselyproportional to its rate of rotation. When a fluorescently labeledtracer-antibody complex is excited by a linearly polarized light, theemitted light remains highly polarized because the fluorophore isconstrained from rotating between the time light is absorbed and thetime light is emitted. When a “free” tracer compound (i.e., a compoundthat is not bound to an antibody) is excited by linearly polarizedlight, its rotation is much faster than the correspondingtracer-antibody conjugate produced in a competitive binding immunoassay.FPIAs are advantageous over RIAs inasmuch as there are no radioactivesubstances requiring special handling and disposal. In addition, FPIAsare homogeneous assays that can be easily and rapidly performed.

In view of the above, a method of determining the presence, amount, orconcentration of analyte (or a fragment thereof) in a test sample isprovided. The method comprises assaying the test sample for an analyte(or a fragment thereof) by an assay (i) employing (i′) at least one ofan antibody, a fragment of an antibody that can bind to an analyte, avariant of an antibody that can bind to an analyte, a fragment of avariant of an antibody that can bind to an analyte, and a DVD-Ig (or afragment, a variant, or a fragment of a variant thereof) that can bindto an analyte, and (ii′) at least one detectable label and (ii)comprising comparing a signal generated by the detectable label as adirect or indirect indication of the presence, amount or concentrationof analyte (or a fragment thereof) in the test sample to a signalgenerated as a direct or indirect indication of the presence, amount orconcentration of analyte (or a fragment thereof) in a control orcalibrator. The calibrator is optionally part of a series ofcalibrators, in which each of the calibrators differs from the othercalibrators by the concentration of analyte.

The method can comprise (i) contacting the test sample with at least onefirst specific binding partner for analyte (or a fragment thereof)selected from the group consisting of an antibody, a fragment of anantibody that can bind to an analyte, a variant of an antibody that canbind to an analyte, a fragment of a variant of an antibody that can bindto an analyte, and a DVD-Ig (or a fragment, a variant, or a fragment ofa variant thereof) that can bind to an analyte so as to form a firstspecific binding partner/analyte (or fragment thereof) complex, (ii)contacting the first specific binding partner/analyte (or fragmentthereof) complex with at least one second specific binding partner foranalyte (or fragment thereof) selected from the group consisting of adetectably labeled anti-analyte antibody, a detectably labeled fragmentof an anti-analyte antibody that can bind to analyte, a detectablylabeled variant of an anti-analyte antibody that can bind to analyte, adetectably labeled fragment of a variant of an anti-analyte antibodythat can bind to analyte, and a detectably labeled DVD-Ig (or afragment, a variant, or a fragment of a variant thereof) so as to form afirst specific binding partner/analyte (or fragment thereof)/secondspecific binding partner complex, and (iii) determining the presence,amount or concentration of analyte in the test sample by detecting ormeasuring the signal generated by the detectable label in the firstspecific binding partner/analyte (or fragment thereof)/second specificbinding partner complex formed in (ii). A method in which at least onefirst specific binding partner for analyte (or a fragment thereof)and/or at least one second specific binding partner for analyte (or afragment thereof) is a DVD-Ig (or a fragment, a variant, or a fragmentof a variant thereof) as described herein can be preferred.

Alternatively, the method can comprise contacting the test sample withat least one first specific binding partner for an IL-1β analyte (or afragment thereof) selected from the group consisting of an antibody, afragment of an antibody that can bind to an analyte, a variant of anantibody that can bind to an analyte, a fragment of a variant of anantibody that can bind to an analyte, and a DVD-Ig (or a fragment, avariant, or a fragment of a variant thereof) and simultaneously orsequentially, in either order, contacting the test sample with at leastone second specific binding partner, which can compete with analyte (ora fragment thereof) for binding to the at least one first specificbinding partner and which is selected from the group consisting of adetectably labeled analyte, a detectably labeled fragment of analytethat can bind to the first specific binding partner, a detectablylabeled variant of analyte that can bind to the first specific bindingpartner, and a detectably labeled fragment of a variant of analyte thatcan bind to the first specific binding partner. Any IL-1β (or a fragmentthereof) present in the test sample and the at least one second specificbinding partner compete with each other to form a first specific bindingpartner/analyte (or fragment thereof) complex and a first specificbinding partner/second specific binding partner complex, respectively.The method further comprises determining the presence, amount orconcentration of analyte in the test sample by detecting or measuringthe signal generated by the detectable label in the first specificbinding partner/second specific binding partner complex formed in (ii),wherein the signal generated by the detectable label in the firstspecific binding partner/second specific binding partner complex isinversely proportional to the amount or concentration of analyte in thetest sample.

The above methods can further comprise diagnosing, prognosticating, orassessing the efficacy of a therapeutic/prophylactic treatment of apatient from whom the test sample was obtained. If the method furthercomprises assessing the efficacy of a therapeutic/prophylactic treatmentof the patient from whom the test sample was obtained, the methodoptionally further comprises modifying the therapeutic/prophylactictreatment of the patient as needed to improve efficacy. The method canbe adapted for use in an automated system or a semi-automated system.

With regard to the methods of assay (and kit therefor), it may bepossible to employ commercially available anti-analyte antibodies ormethods for production of anti-analyte as described in the literature.Commercial supplies of various antibodies include, but are not limitedto, Santa Cruz Biotechnology Inc. (Santa Cruz, Calif.), GenWay Biotech,Inc. (San Diego, Calif.), and R&D Systems (RDS; Minneapolis, Minn.).

Generally, a predetermined level can be employed as a benchmark againstwhich to assess results obtained upon assaying a test sample for analyteor a fragment thereof, e.g., for detecting disease or risk of disease.Generally, in making such a comparison, the predetermined level isobtained by running a particular assay a sufficient number of times andunder appropriate conditions such that a linkage or association ofanalyte presence, amount or concentration with a particular stage orendpoint of a disease, disorder or condition or with particular clinicalindicia can be made. Typically, the predetermined level is obtained withassays of reference subjects (or populations of subjects). The analytemeasured can include fragments thereof, degradation products thereof,and/or enzymatic cleavage products thereof.

In particular, with respect to a predetermined level as employed formonitoring disease progression and/or treatment, the amount orconcentration of analyte or a fragment thereof may be “unchanged,”“favorable” (or “favorably altered”), or “unfavorable” (or “unfavorablyaltered”). “Elevated” or “increased” refers to an amount or aconcentration in a test sample that is higher than a typical or normallevel or range (e.g., predetermined level), or is higher than anotherreference level or range (e.g., earlier or baseline sample). The term“lowered” or “reduced” refers to an amount or a concentration in a testsample that is lower than a typical or normal level or range (e.g.,predetermined level), or is lower than another reference level or range(e.g., earlier or baseline sample). The term “altered” refers to anamount or a concentration in a sample that is altered (increased ordecreased) over a typical or normal level or range (e.g., predeterminedlevel), or over another reference level or range (e.g., earlier orbaseline sample).

The typical or normal level or range for analyte is defined inaccordance with standard practice. Because the levels of analyte in someinstances will be very low, a so-called altered level or alteration canbe considered to have occurred when there is any net change as comparedto the typical or normal level or range, or reference level or range,that cannot be explained by experimental error or sample variation.Thus, the level measured in a particular sample will be compared withthe level or range of levels determined in similar samples from aso-called normal subject. In this context, a “normal subject” is anindividual with no detectable disease, for example, and a “normal”(sometimes termed “control”) patient or population is/are one(s) thatexhibit(s) no detectable disease, respectively, for example.Furthermore, given that analyte is not routinely found at a high levelin the majority of the human population, a “normal subject” can beconsidered an individual with no substantial detectable increased orelevated amount or concentration of analyte, and a “normal” (sometimestermed “control”) patient or population is/are one(s) that exhibit(s) nosubstantial detectable increased or elevated amount or concentration ofanalyte. An “apparently normal subject” is one in which analyte has notyet been or currently is being assessed. The level of an analyte is saidto be “elevated” when the analyte is normally undetectable (e.g., thenormal level is zero, or within a range of from about 25 to about 75percentiles of normal populations), but is detected in a test sample, aswell as when the analyte is present in the test sample at a higher thannormal level. Thus, inter alia, the disclosure provides a method ofscreening for a subject having, or at risk of having, a particulardisease, disorder, or condition. The method of assay can also involvethe assay of other markers and the like.

Accordingly, the methods described herein also can be used to determinewhether or not a subject has or is at risk of developing a givendisease, disorder or condition. Specifically, such a method can comprisethe steps of:

(a) determining the concentration or amount in a test sample from asubject of IL-1β (or a fragment thereof) (e.g., using the methodsdescribed herein, or methods known in the art); and

(b) comparing the concentration or amount of IL-1β (or a fragmentthereof) determined in step (a) with a predetermined level, wherein, ifthe concentration or amount of analyte determined in step (a) isfavorable with respect to a predetermined level, then the subject isdetermined not to have or be at risk for a given disease, disorder orcondition. However, if the concentration or amount of IL-1β determinedin step (a) is unfavorable with respect to the predetermined level, thenthe subject is determined to have or be at risk for a given disease,disorder or condition.

Additionally, provided herein is method of monitoring the progression ofdisease in a subject. Optimally the method comprising the steps of:

(a) determining the concentration or amount in a test sample from asubject of IL-1β;

(b) determining the concentration or amount in a later test sample fromthe subject of IL-1β; and

(c) comparing the concentration or amount of analyte as determined instep (b) with the concentration or amount of IL-1β determined in step(a), wherein if the concentration or amount determined in step (b) isunchanged or is unfavorable when compared to the concentration or amountof IL-1β determined in step (a), then the disease in the subject isdetermined to have continued, progressed or worsened. By comparison, ifthe concentration or amount of IL-1β as determined in step (b) isfavorable when compared to the concentration or amount of IL-1β asdetermined in step (a), then the disease in the subject is determined tohave discontinued, regressed or improved.

Optionally, the method further comprises comparing the concentration oramount of IL-1β analyte as determined in step (b), for example, with apredetermined level. Further, optionally the method comprises treatingthe subject with one or more pharmaceutical compositions for a period oftime if the comparison shows that the concentration or amount of analyteas determined in step (b), for example, is unfavorably altered withrespect to the predetermined level.

Still further, the methods can be used to monitor treatment in a subjectreceiving treatment with one or more pharmaceutical compositions.Specifically, such methods involve providing a first test sample from asubject before the subject has been administered one or morepharmaceutical compositions. Next, the concentration or amount in afirst test sample from a subject of IL-1β is determined (e.g., using themethods described herein or as known in the art). After theconcentration or amount of IL-1β is determined, optionally theconcentration or amount of IL-1β is then compared with a predeterminedlevel. If the concentration or amount of IL-1β as determined in thefirst test sample is lower than the predetermined level, then thesubject is not treated with one or more pharmaceutical compositions.However, if the concentration or amount of IL-1β as determined in thefirst test sample is higher than the predetermined level, then thesubject is treated with one or more pharmaceutical compositions for aperiod of time. The period of time that the subject is treated with theone or more pharmaceutical compositions can be determined by one skilledin the art (for example, the period of time can be from about seven (7)days to about two years, preferably from about fourteen (14) days toabout one (1) year).

During the course of treatment with the one or more pharmaceuticalcompositions, second and subsequent test samples are then obtained fromthe subject. The number of test samples and the time in which said testsamples are obtained from the subject are not critical. For example, asecond test sample could be obtained seven (7) days after the subject isfirst administered the one or more pharmaceutical compositions, a thirdtest sample could be obtained two (2) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fourth testsample could be obtained three (3) weeks after the subject is firstadministered the one or more pharmaceutical compositions, a fifth testsample could be obtained four (4) weeks after the subject is firstadministered the one or more pharmaceutical compositions, etc.

After each second or subsequent test sample is obtained from thesubject, the concentration or amount of IL-1β analyte is determined inthe second or subsequent test sample is determined (e.g., using themethods described herein or as known in the art). The concentration oramount of IL-1β as determined in each of the second and subsequent testsamples is then compared with the concentration or amount of analyte asdetermined in the first test sample (e.g., the test sample that wasoriginally optionally compared to the predetermined level). If theconcentration or amount of IL-1β as determined in step (c) is favorablewhen compared to the concentration or amount of analyte as determined instep (a), then the disease in the subject is determined to havediscontinued, regressed or improved, and the subject should continue tobe administered the one or pharmaceutical compositions of step (b).However, if the concentration or amount determined in step (c) isunchanged or is unfavorable when compared to the concentration or amountof analyte as determined in step (a), then the disease in the subject isdetermined to have continued, progressed or worsened, and the subjectshould be treated with a higher concentration of the one or morepharmaceutical compositions administered to the subject in step (b) orthe subject should be treated with one or more pharmaceuticalcompositions that are different from the one or more pharmaceuticalcompositions administered to the subject in step (b). Specifically, thesubject can be treated with one or more pharmaceutical compositions thatare different from the one or more pharmaceutical compositions that thesubject had previously received to decrease or lower said subject'sanalyte level.

Generally, for assays in which repeat testing may be done (e.g.,monitoring disease progression and/or response to treatment), a secondor subsequent test sample is obtained at a period in time after thefirst test sample has been obtained from the subject. Specifically, asecond test sample from the subject can be obtained minutes, hours,days, weeks or years after the first test sample has been obtained fromthe subject. For example, the second test sample can be obtained fromthe subject at a time period of about 1 minute, about 5 minutes, about10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days,about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks,about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years after the first test sample from the subject is obtained.

When used to monitor disease progression, the above assay can be used tomonitor the progression of disease in subjects suffering from acuteconditions. Acute conditions, also known as critical care conditions,refer to acute, life-threatening diseases or other critical medicalconditions involving, for example, the cardiovascular system orexcretory system. Typically, critical care conditions refer to thoseconditions requiring acute medical intervention in a hospital-basedsetting (including, but not limited to, the emergency room, intensivecare unit, trauma center, or other emergent care setting) oradministration by a paramedic or other field-based medical personnel.For critical care conditions, repeat monitoring is generally done withina shorter time frame, namely, minutes, hours or days (e.g., about 1minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3days, about 4 days, about 5 days, about 6 days or about 7 days), and theinitial assay likewise is generally done within a shorter timeframe,e.g., about minutes, hours or days of the onset of the disease orcondition.

The assays also can be used to monitor the progression of disease insubjects suffering from chronic or non-acute conditions. Non-criticalcare or, non-acute conditions, refers to conditions other than acute,life-threatening disease or other critical medical conditions involving,for example, the cardiovascular system and/or excretory system.Typically, non-acute conditions include those of longer-term or chronicduration. For non-acute conditions, repeat monitoring generally is donewith a longer timeframe, e.g., hours, days, weeks, months or years(e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours,about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours,about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks,about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks,about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31 weeks,about 32 weeks, about 33 weeks, about 34 weeks, about 35 weeks, about 36weeks, about 37 weeks, about 38 weeks, about 39 weeks, about 40 weeks,about 41 weeks, about 42 weeks, about 43 weeks, about 44 weeks, about 45weeks, about 46 weeks, about 47 weeks, about 48 weeks, about 49 weeks,about 50 weeks, about 51 weeks, about 52 weeks, about 1.5 years, about 2years, about 2.5 years, about 3.0 years, about 3.5 years, about 4.0years, about 4.5 years, about 5.0 years, about 5.5. years, about 6.0years, about 6.5 years, about 7.0 years, about 7.5 years, about 8.0years, about 8.5 years, about 9.0 years, about 9.5 years or about 10.0years), and the initial assay likewise generally is done within a longertime frame, e.g., about hours, days, months or years of the onset of thedisease or condition.

Furthermore, the above assays can be performed using a first test sampleobtained from a subject where the first test sample is obtained from onesource, such as urine, serum or plasma. Optionally, the above assays canthen be repeated using a second test sample obtained from the subjectwhere the second test sample is obtained from another source. Forexample, if the first test sample was obtained from urine, the secondtest sample can be obtained from serum or plasma. The results obtainedfrom the assays using the first test sample and the second test samplecan be compared. The comparison can be used to assess the status of adisease or condition in the subject.

Moreover, the present disclosure also relates to methods of determiningwhether a subject predisposed to or suffering from a given disease,disorder or condition will benefit from treatment. In particular, thedisclosure relates to analyte companion diagnostic methods and products.Thus, the method of “monitoring the treatment of disease in a subject”as described herein further optimally also can encompass selecting oridentifying candidates for therapy.

Thus, in particular embodiments, the disclosure also provides a methodof determining whether a subject having, or at risk for, a givendisease, disorder or condition is a candidate for therapy. Generally,the subject is one who has experienced some symptom of a given disease,disorder or condition or who has actually been diagnosed as having, orbeing at risk for, a given disease, disorder or condition, and/or whodemonstrates an unfavorable concentration or amount of analyte or afragment thereof, as described herein.

The method optionally comprises an assay as described herein, whereIL-1β is assessed before and following treatment of a subject with oneor more pharmaceutical compositions (e.g., particularly with apharmaceutical related to a mechanism of action involving analyte), withimmunosuppressive therapy, or by immunoabsorption therapy, or whereanalyte is assessed following such treatment and the concentration orthe amount of analyte is compared against a predetermined level. Anunfavorable concentration of amount of IL-1β observed followingtreatment confirms that the subject will not benefit from receivingfurther or continued treatment, whereas a favorable concentration oramount of analyte observed following treatment confirms that the subjectwill benefit from receiving further or continued treatment. Thisconfirmation assists with management of clinical studies, and provisionof improved patient care.

It goes without saying that, while certain embodiments herein areadvantageous when employed to assess a given disease, disorder orcondition as discussed herein, the assays and kits can be employed toassess analyte in other diseases, disorders and conditions. The methodof assay can also involve the assay of other markers and the like.

The method of assay also can be used to identify a compound thatameliorates a given disease, disorder or condition. For example, a cellthat expresses analyte can be contacted with a candidate compound. Thelevel of expression of analyte in the cell contacted with the compoundcan be compared to that in a control cell using the method of assaydescribed herein.

II. Kits

A kit for assaying a test sample for the presence, amount orconcentration of an analyte (or a fragment thereof) in a test sample isalso provided. The kit comprises at least one component for assaying thetest sample for IL-1β (or a fragment thereof) and instructions forassaying the test sample for the analyte (or a fragment thereof). The atleast one component for assaying the test sample for the analyte (or afragment thereof) can include a composition comprising an anti-IL-1βbinding protein, such as a monoclonal antibody or DVD-Ig (or a fragment,a variant, or a fragment of a variant thereof), as described herein andwhich is optionally immobilized on a solid phase.

The kit can comprise at least one component for assaying the test samplefor an IL-1β analyte by immunoassay, e.g., chemiluminescentmicroparticle immunoassay, and instructions for assaying the test samplefor an IL-1β analyte by immunoassay, e.g., chemiluminescentmicroparticle immunoassay. For example, the kit can comprise at leastone specific binding partner for IL-1β, such as an anti-IL-1βmonoclonal/polyclonal antibody (or a fragment thereof that can bind tothe IL-1β analyte, a variant thereof that can bind to the analyte, or afragment of a variant that can bind to the analyte) or an anti-IL-1βDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof),either of which can be detectably labeled. Alternatively oradditionally, the kit can comprise detectably labeled IL-1β analyte (ora fragment thereof that can bind to an anti-analyte,monoclonal/polyclonal antibody or an anti-analyte DVD-Ig (or a fragment,a variant, or a fragment of a variant thereof)), which can compete withany analyte in a test sample for binding to an anti-analytemonoclonal/polyclonal antibody (or a fragment thereof that can bind tothe analyte, a variant thereof that can bind to the analyte, or afragment of a variant that can bind to the analyte) or an anti-analyteDVD-Ig (or a fragment, a variant, or a fragment of a variant thereof),either of which can be immobilized on a solid support. The kit cancomprise a calibrator or control, e.g., isolated or purified analyte.The kit can comprise at least one container (e.g., tube, microtiterplates or strips, which can be already coated with a first specificbinding partner, for example) for conducting the assay, and/or a buffer,such as an assay buffer or a wash buffer, either one of which can beprovided as a concentrated solution, a substrate solution for thedetectable label (e.g., an enzymatic label), or a stop solution.Preferably, the kit comprises all components, i.e., reagents, standards,buffers, diluents, etc., which are necessary to perform the assay. Theinstructions can be in paper form or computer-readable form, such as adisk, CD, DVD, or the like.

Any binding protein, such as an anti-IL-1β binding protein or ananti-analyte DVD-Ig, or tracer can incorporate a detectable label asdescribed herein, such as a fluorophore, a radioactive moiety, anenzyme, a biotin/avidin label, a chromophore, a chemiluminescent label,or the like, or the kit can include reagents for carrying out detectablelabeling. The antibodies, calibrators and/or controls can be provided inseparate containers or pre-dispensed into an appropriate assay format,for example, into microtiter plates.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the immunoassay kit reagents, and the standardization ofassays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, enzyme substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers as necessary, e.g., a microtiter plate. The kit can furtherinclude containers for holding or storing a sample (e.g., a container orcartridge for a urine sample). Where appropriate, the kit optionallyalso can contain reaction vessels, mixing vessels, and other componentsthat facilitate the preparation of reagents or the test sample. The kitcan also include one or more instruments for assisting with obtaining atest sample, such as a syringe, pipette, forceps, measured spoon, or thelike.

If the detectable label is at least one acridinium compound, the kit cancomprise at least one acridinium-9-carboxamide, at least oneacridinium-9-carboxylate aryl ester, or any combination thereof. If thedetectable label is at least one acridinium compound, the kit also cancomprise a source of hydrogen peroxide, such as a buffer, a solution,and/or at least one basic solution. If desired, the kit can contain asolid phase, such as a magnetic particle, bead, test tube, microtiterplate, cuvette, membrane, scaffolding molecule, film, filter paper, discor chip.

III. Adaptation of Kit and Method

The kit (or components thereof), as well as the method of determiningthe presence, amount or concentration of an analyte in a test sample byan assay, such as an immunoassay as described herein, can be adapted foruse in a variety of automated and semi-automated systems (includingthose wherein the solid phase comprises a microparticle), as described,e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as commerciallymarketed, e.g., by Abbott Laboratories (Abbott Park, Ill.) asARCHITECT®.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., an anti-analyte,monoclonal/polyclonal antibody (or a fragment thereof, a variantthereof, or a fragment of a variant thereof) or an anti-analyte DVD-Ig(or a fragment thereof, a variant thereof, or a fragment of a variantthereof) is attached; either way, sandwich formation and analytereactivity can be impacted), and the length and timing of the capture,detection and/or any optional wash steps. Whereas a non-automatedformat, such as an ELISA, may require a relatively longer incubationtime with sample and capture reagent (e.g., about 2 hours), an automatedor semi-automated format (e.g., ARCHITECT®, Abbott Laboratories) mayhave a relatively shorter incubation time (e.g., approximately 18minutes for ARCHITECT®). Similarly, whereas a non-automated format, suchas an ELISA, may incubate a detection antibody, such as the conjugatereagent, for a relatively longer incubation time (e.g., about 2 hours),an automated or semi-automated format (e.g., ARCHITECT®) may have arelatively shorter incubation time (e.g., approximately 4 minutes forthe ARCHITECT®).

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404), PRISM®,EIA (bead), and Quantum™ II, as well as other platforms. Additionally,the assays, kits and kit components can be employed in other formats,for example, on electrochemical or other hand-held or point-of-careassay systems. The present disclosure is, for example, applicable to thecommercial Abbott Point of Care (i-STAT®, Abbott Laboratories)electrochemical immunoassay system that performs sandwich immunoassays.Immunosensors and their methods of manufacture and operation insingle-use test devices are described, for example in, U.S. Pat. No.5,063,081, US Publication No. 2003/0170881, US Publication No.2004/0018577, US Publication No. 2005/0054078, and US Publication No.2006/0160164.

In particular, with regard to the adaptation of an analyte assay to theI-STAT® system, the following configuration is preferred. Amicrofabricated silicon chip is manufactured with a pair of goldamperometric working electrodes and a silver-silver chloride referenceelectrode. On one of the working electrodes, polystyrene beads (0.2 mmdiameter) with immobilized anti-analyte, monoclonal/polyclonal antibody(or a fragment thereof, a variant thereof, or a fragment of a variantthereof) or anti-analyte DVD-Ig (or a fragment thereof, a variantthereof, or a fragment of a variant thereof), are adhered to a polymercoating of patterned polyvinyl alcohol over the electrode. This chip isassembled into an I-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the wall of the sample-holding chamber ofthe cartridge there is a layer comprising a specific binding partner foran analyte, such as an anti-analyte, monoclonal/polyclonal antibody (ora fragment thereof, a variant thereof, or a fragment of a variantthereof that can bind the analyte) or an anti-analyte DVD-Ig (or afragment thereof, a variant thereof, or a fragment of a variant thereofthat can bind the analyte), either of which can be detectably labeled.Within the fluid pouch of the cartridge is an aqueous reagent thatincludes p-aminophenol phosphate.

In operation, a sample suspected of containing an analyte is added tothe holding chamber of the test cartridge, and the cartridge is insertedinto the I-STAT® reader. After the specific binding partner for ananalyte has dissolved into the sample, a pump element within thecartridge forces the sample into a conduit containing the chip. Here itis oscillated to promote formation of the sandwich. In the penultimatestep of the assay, fluid is forced out of the pouch and into the conduitto wash the sample off the chip and into a waste chamber. In the finalstep of the assay, the alkaline phosphatase label reacts withp-aminophenol phosphate to cleave the phosphate group and permit theliberated p-aminophenol to be electrochemically oxidized at the workingelectrode. Based on the measured current, the reader is able tocalculate the amount of analyte in the sample by means of an embeddedalgorithm and factory-determined calibration curve.

It further goes without saying that the methods and kits as describedherein necessarily encompass other reagents and methods for carrying outthe immunoassay. For instance, encompassed are various buffers such asare known in the art and/or which can be readily prepared or optimizedto be employed, e.g., for washing, as a conjugate diluent, microparticlediluent, and/or as a calibrator diluent. An exemplary conjugate diluentis ARCHITECT® conjugate diluent employed in certain kits (AbbottLaboratories, Abbott Park, Ill.) and containing2-(N-morpholino)ethanesulfonic acid (MES), a salt, a protein blocker, anantimicrobial agent, and a detergent. An exemplary calibrator diluent isARCHITECT® human calibrator diluent employed in certain kits (AbbottLaboratories, Abbott Park, Ill.), which comprises a buffer containingMES, other salt, a protein blocker, and an antimicrobial agent.Additionally, as described in U.S. Ser. No. 12/650,241 (US PublicationNo. 2010/0167301; see, also PCT Publication No. WO 2010/078443),improved signal generation may be obtained, e.g., in an 1-Stat cartridgeformat, using a nucleic acid sequence linked to the signal antibody as asignal amplifier.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included for purposes of illustration only and are not intended tobe limiting of the invention.

EXEMPLIFICATIONS Example 1 Generation of Affinity-Matured HumanizedIL-1β Antibodies from Clone E26

Table 6 provides the amino acid sequences of the VH and VL of thehumanized mouse E26 antibody (GlaxoSmithKline, PCT Publication No. WO95/01997). The amino acid residues of individual CDRs of each VH and VLsequence are indicated in bold.

TABLE 6 Amino Acid Sequences of VH and VL Regions of Humanized E26 AntibodySEQ ID Protein Sequence NO: region 123456789012345678901234567890 58VH E26 EVQLVESGGGVVQPGRSLRLSCSSSGFIFS SYDMSWVRQAPGKGLEWVAYISSGGGGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPED TGVYFCARGGVTKGYFDVWGQGTPVTVSS VH E26Residues 31-35 SYDMS CDR-H1 of SEQ-ID NO: 58 VH E26 Residues 50-66YISSGGGGTYYPDTVKG CDR-H2 of SEQ-ID NO: 58 VH E26 Residues 99-108GGVTKGYFDV CDR-H3 of SEQ-ID NO: 58 59 VL E26DIQMTQSPSSLSASVGDRVTITCRASGNIH NYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH FWSIPYTFGQGTKLQIT VL E26 Residues 24-34RASGNIHNYLT CDR-L1 of SEQ-ID NO: 59 VL E26 Residues 50-56 NAKTLAD CDR-L2of SEQ-ID NO: 59 VL E26 Residues 89-97 QHFWSIPYT CDR-L3 of SEQ-ID NO: 59

Affinity matured humanized mouse E26 antibodies were obtained asfollows. One light chain library was constructed to contain limitedmutagenesis at the following residues: CDRL1: 30, 31, 32; CDRL2: 50, 53,55, 56; CDRL3: 92, 93, 94, 96, and 97 (Kabat numbering). Two heavy chainlibraries were made to contain limited mutagenesis in CDRH1 and CDRH2 atresidues 31, 33, 50, 52a, 55, 56, 57, 58, and 60 (Kabat numbering) or inCDRH3 at residues 95, 96, 97, 98, 99, 100, 100a, 100b, and 102. Theheavy chain libraries also contained binary diversities at residues23(A/S), 24(A/S), 62(T/S), 84(P/A), 88(G/A), 91(F/Y), and 108(P/L) toallow for framework germ-lining during library selections. All threelibraries were selected separately by decreasing concentrations ofcynomolgus (cyno) IL-113. All mutated CDR sequences were then combinedinto one library having mutations in the VH CDRs only and anotherlibrary having mutations in all six CDRs. These two combined librarieswere subjected to more stringent selection conditions with human andcyno IL-1β before individual antibodies were identified and expressed asIgG proteins for further characterization.

Table 7 provides a list of amino acid sequences of VH region of affinitymatured IL-1β antibodies derived from humanized E26. The amino acidresidues of individual CDRs of each VH sequence are indicated in bold.

TABLE 7  Amino Acid Sequences of Affinity Matured E26 VH VariantsSequence Clone SEQ ID NO:  1234567890123456789012345678901234567890E26 #1 60 EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDEWGQGTPVTVSS E26 #11 61EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS E26 #35 62EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS E26 #37 63EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS J318 #2 64EVQLVESGGGVVQPGRSLRLSCAASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS J318 #12 65EVQLVESGGGVVQPGRSLRLSCAASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS J318 #13 66EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS J348S2-10 67EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTPVTVSS J348S2-85 68EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTLVTVSS J348S2-1 69EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDEWGQGTPVTVSS J348S2-37 70EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS J348S2-49 71EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDEWGQGTPVTVSS J348S2-56 72EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDEWGQGTPVTVSS J348S2-25 73EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS J348S2-45 74EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS J348S2-94 75EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS J348S2-34 76EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRVEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS J348S2-58 77EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS J348S2-61 78EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDLWGQGTPVTVS J348S2-80 79EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYFCARGGVYKGYFDLWGQGTPVTVSS J348S2-96 80EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYFCARGGVYKGYFDVWGQGTPVTVSS J348S2-90 81EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTVSRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTPVTVSS J348S2-21 82EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDEWGQGTPVTVSS J348S2-39 83EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDEWGQGTPVTVSS J348S2-53 84EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVYKGYFDEWGQGTPVTVSS J348S2-74 85EVQLVESGGGVVQPGRSLRLSCSSSGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYFCARGGVYKGYFDEWGQGTPVTVSS J348S2-30 86EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTPVTVSS J348S2-73 87EVQLVESGGGVVQPGRSLRLSCSSSGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDMWGQGTPVTVSS J348S2-12 88EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-92 89EVQLVESGGGVVQPGRSLRLSCASSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-14 90EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-33 91EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-2 92EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCVRGGVYKGYFDQWGQGTLVTVSS J348S2-65 93EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCVRGGVYKGYFDQWGQGTLVTVSS J348S2-20 94EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-54 95EVQLVESGGGVVQPGRSLRLSCSSSGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVGS J348S2-13 96EVQLVESGGGVVQPGRSLRLSCSSSGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-17 97EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-44 98EVQLVEGGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-47 99EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-48 100EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-22 101EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-42 102EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS J348S2-84 381EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTPVTVSS gi10 103EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDEWGQGTPVTVSS gi15 104EVQLVESGGGVVQPGRGLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSRNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDYWGQGTPVTVSS gi68 105EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYVSHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDYWGQGTPVTVSS gi80 106EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDYWGQGTPVTVSS gi5 107EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDEWGQGTPVTVSS gi49 108EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi78 109EVQLVESGGGVVQPGRSLRLGCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi4 110EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi66 111EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMNSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi77 112EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi19 113EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTAKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi33 114EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYGMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi58 115EVQLVESGGDVVQPGRSLRLSCSAGGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi79 116EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi37 117EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMGWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi9 118EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVHQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi1 119EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi2 120EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi38 121EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVYKGYFDQWGQGTPVTVSS gi74 122EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYFCARGGVYKGYFDVWGQGTPVTVSS gi27 123EVQLVESGGGVVQPGRSLRLSCSSSGFIFSSYDMSWVRQAPGKGLEWVAYISSGGGGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS gi64 124EVQLVESGGGVVQPGRSLRLSCSSSGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi85 125EVQLVESGGGVVQPGRSLRLSCSSSGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDVWGQGTPVTVSL gi46 126EVQLVESGGGVVQPGRSLRLSCSSSGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCVRGGVYKGYFDVWGQGTPVTVSS gi35 127EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi45 128EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSPRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi90 129EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi11 130EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi21 131EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSRVRLAPGKGLEWVAYISHGGAGTYHPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi59 132EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS gi91 133EVQLVESGGDVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYIGHGGAGTYYPDTVKGRFTISRNNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS gi60 134EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDEWGQGTLVTVSS gi36 135EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDLWGQGTPVTVSS gi12 136EVQLVESGGGVVQPGRSLRLSCASSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi55 137EVQLVESGGGVVQPGRSLRLSCASSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi13 138EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi11 139EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGPEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDEWGQGTPVTVSS gi16 140EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGCFDVWGQGTPVTVSS gi39 141EVQLVESGGGVVQPGRSLRLSCSSSGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS gi24 142EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAEDTGVYFCARGGVYKGYFDEWGQGTPVTVSS gi67 143EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVYKGYFDEWGQGTPVTVSS gi65 144EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVYKGYFDVWGQGTPVTVSS gi25 145EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDTVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi20 146EVQLVESGGGVVQPGRSLRLSCSASGFIFSKYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS gi72 147EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTLVTVSS gi84 148EVQLVESGGGVVQPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYYCARGGVYKGYFDVWGQGTPVTVSS

Table 8 provides a list of amino acid sequences of VL regions ofaffinity matured humanized IL-113 antibodies derived from E26. The aminoacid residues of individual CDRs of each VL sequence are indicated inbold. The N-terminal D (Asp) to G (Gly) mutation seen in some of theaffinity matured VL sequences in Table 8 below was most likely a resultof unintended mutagenesis that occurred during polymerase chain reaction(PCR) carried out during library construction. The N-terminal G residuecould be removed without consequence when these regions were used in theconstruction of IgG molecules.

TABLE 8  Amino Acid Sequences of Affinity Matured E26 VL Variants SEQ IDSequence Clone NO: 1234567890123456789012345678901234567890 E26 #1 149DIQMTQSPSSLSASVGDRVTITCRASGNIYGWLAWYQQTPGKAPKLLIYQAKTLMDGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWNIPATFGQGTKLQITE26 #37 150 DIQMTQSPSSLSASVGDRVTITCRASGNIYTYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQITJ348S2-10 151 GIQMTQSPSSLSASVGDRVTITCRASGNIYQYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-84 152 GIQMTQSPSSLSASVGDRVTITCRASGNIYQYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-2 153 DIQMTQSPSSLSASVGDRVTITCRASGNIYEYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-73 154 GIQMAQSPSSLSASVGDRVTITCRASGNIYEYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-13 155 GIQMTQSPSSLSASVGDRVTITCRASGNIYTYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWSLPYTFGQGTKLQITJ348S2-18 156 GIQMTQSPSSLSASVGDRVTITCRASGNIYHYLTWYQQTPGKAPKLLIYDAKTLAEGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWSLPYTFGQGTKLQITJ348S2-24 157 GIQMTQSPSSLSASVGDRVTITCRASGNIYHYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWSLPYTFGQGTKLQITJ348S2-22 158 GIQMTQSPSSLSASVGDRVTITCRASGNIYHYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSCSGTDYTFTISSLQP EDIATYYCQHFWSLPYTFGQGTKLQITJ348S2-12 159 GIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQITJ348S2-44 160 GIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSSSGTDYTLTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQITJ348S2-37 161 GIQMTQSPSSLSASVGDRVTITCRASGNIYTYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQITJ348S2-74 162 GIQMTQSPSSLSASVGDRVTITCRASGNIYQYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQITJ348S2-57 163 GIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQITJ348S2-16 164 GIQMTQSPSSLSASVGDRVTITCRASGNIYDYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSCSGTDYTFTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQITJ348S2-33 165 GIQMTQSPSSLSASVGDRVTITCRASGNIYDYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSCSGTDYTFTISSLQP EDIATYYCQHFWSLPYTFGQGTKLQITJ348S2-20 166 GIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSCSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-15 167 GIQMTQSPSSLSASVGDRVTITCRASGNIYKYLTWYQQTPGKAPKLLIYDAKTLADGVPSRFSGSVSGTDYTFTISSLQP EDIATYYCQHFWRLPYTFGQGTKLQITJ348S2-48 168 GIQMTQSPSSLSASVGDRVTITCRASGNIYKYLTWYQQTPGKAPKLLIYDAKTLADGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWRLPYTFGQGTKLQITJ348S2-32 169 GIQMTQSPSSLSASVGDRVTITCRASGNIYHYLTWYQQTPGKAPKLLIYDAKNLADGVPSRFSGSCSGTDYTFTISSLQP EDIATYYCQHFWSLPYTFGQGTKLQITJ348S2-49 170 GIQMTQSPSSLSASVGDRVTITCRASGNIYHYLTWYQQTPGKAPKLLIYDAKTLADGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWSLPYTFGQGTKLQITJ348S2-78 171 GIQMTQSPSSLSASVGDRVTITCRASGNIYQYLTWYQQTPGKAPKLLIYDAKILADGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-96 172 GIQMTQSPSSLSASVGDRVTITCRASGNIYHYLTWYQQTPGKAPKLLIYDAKILADGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-1 173 DIQMTQSPSSLSASVGDRVTITCRASGNIYGWLAWYQQTPGKAPKLLIYQAKTLMDGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWNIPATFGQGTKLQITJ348S2-25 174 DIQMTQSPSSLSASVGDRVTITCRASGNIYTYLNWYQQTPGKAPKLLIYDAKTLADGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWRLPYTFGQGTKLQITJ348S2-34 175 DIQMTQSPSSLSASVGDRVTITCRASGNIYTYLNWYQQTPGKAPKLLIYNAKELAEGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWRLPYTFGQGTKLQITJ348S2-65 176 DIQMTQSPSSLSASVGDRVTITCRASGNIYTYLNWYQQTPGKAPKLLIYNAKSLADGVPSRFSGSCSGTDYTFTISSLQP EDIATYYCQHFWRIPYTFGQGTKLQITJ348S2-90 177 DIQMTQSPSSLSASVGDRVTITCRASGNIYTYLTWYQQTPGKAPKLLIYDAKTLADGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-56 178 DIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKNLADGVPSRFSGSVSGTDYTFTISSLQP EDIATYYCQHFWRLPYTFGQGTKLQITJ348S2-61 179 DIQMTQSPSSLSASVGDRVTITCRASGNIWHYLTWYQQTPGKAPKLLIYDAKTLADGVPSRFSGSSSGTDYTLTISSLQP EDIATYYCQHFWRLPYTFGQGTKLQITJ348S2-80 180 DIQMTQSPSSLSASVGDRVTITCRASGNIYHYLTWYQQTPGKAPKLLIYNAKTLASGVPSRFSGSSSGTDYTLTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-94 181 DIQMTQSPSSLSASVGDRVTITCRASGNIYDYLTWYQQTPGKAPKLLIYNAKILADGVPSRFSGSGSGTDYTLTISSLQP EDIATYYCQHFWMLPYTFGQGTKLQITJ348S2-14 182 GIQMTQSPSSLSASVGDRVTITCRASGNIYTYLTWYQQIPGKAPKLLIYDAKTLAEGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWKIPYTFGQGTKLQITJ348S2-58 183 DIQMTQSPSSLSASVGDRVTITCRASGNIYHYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSCSGADYTFTISSLQP EDIATYYCQQFWKIPYTFGQGTKLQITJ348S2-95 184 DIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSDSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-42 185 GIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKTLAAGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-45 186 GIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWKLPYTFGQGTKLQITJ348S2-17 187 GIQMTQSPSSLSASVGDRVTITCRASGNIYNYLTWYQQTPGKAPKLLIYNAKTLEEGVPSRFSGSSSGTDYTFTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQITJ348S2-53 188 GIQMTQSPSSLSASVGDRVTITCRASGNIYGYLTWYQQTPGKAPKLLIYNAKTLEEGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWTLPYNFGQGTKLQITJ348S2-47 189 GIQMTQSPSSLSASVGDRVTITCRASGNIYDYLTWYQQTPGKAPKLLIYNAKTLAEGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWTLPYTFGQGTKLQIT

The sequences of the individual CDRs of the VH and VL regions of theaffinity matured IL-1β antibodies from humanized E26 in the above tablescan be aligned to provide consensus CDR sequences such as those in Table9.

TABLE 9 Consensus Sequence for Affinity Matured VH and VL Sequences CDRSequence region Identifier Consensus Sequence CDR-H1 SEQ ID X₁ X₂ X₃ X₄X₅ NO: 190 S Y D M S K R CDR-H2 SEQ ID X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ NO: 191 Y I S S G G G G T Y Y P D T V K G VH A S A CDR-H3 SEQ ID X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ NO: 192 G G V T K GY F D V Y C E L M Q Y CDR-L1 SEQ ID X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ X₁₀ X₁₁  NO: 193 R A S G N I H N Y L T Y G W A W T N Q E H D K CDR-L2 SEQ ID X₁X₂ X₃ X₄ X₅ X₆ X₇ NO: 194 N A K T L A D Q N M E D I E S E A S CDR-L3SEQ ID X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈ X₉ NO: 195 Q H F W S I P Y T Q N L A I TN K R M

The sequences in Table 10 were converted into IgG for furthercharacterization. Clone E26.13 was mutated in the J-region of thevariable heavy and light region called E26.13 JM VH and E26.13 JM VL,respectively, to remove non-germline framework mutations. The amino acidresidues of individual CDRs are indicated in bold.

TABLE 10 Amino Acid Sequences of Affinity Matured E26 VH and VL VariantsSEQ ID Sequence NO: Protein Region 123456789012345678901234567890 196E26.1 EVQLVESGGGVVQPGRSLRLSCSASGFIFS VH KYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRAED TGVYYCARGGVYKGYFDEWGQGTPVTVSS E26.1CDR-H1  Residues KYDMS VH 31-35 of SEQ ID NO: 196 E26.1 CDR-H2 ResiduesYISHGGAGTYYPDSVKG VH 50-66 of SEQ ID NO: 196 E26.1 CDR-H3 ResiduesGGVYKGYFDE VH 99-108 of SEQ ID NO: 196 197 E26.1DIQMTQSPSSLSASVGDRVTITCRASGNIY VL GWLAWYQQTPGKAPKLLIYQAKTLMDGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH FWNIPATFGQGTKLQIT E26.1 CDR-L1 ResiduesRASGNIYGWLA VL 24-34 of SEQ ID NO: 197 E26.1 CDR-L2 Residues QAKTLMD VL50-56 of SEQ ID NO: 197 E26.1 CDR-L3 Residues QHFWNIPAT VL 89-97 ofSEQ ID NO: 197 198 E26.2 EVQLVESGGGVVQPGRSLRLSCAASGFIFS VHKYDMSWVRQAPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS E26.2 CDR-H1 Residues KYDMS VH 31-35 ofSEQ ID NO: 198 E26.2 CDR-H2 Residues YISHGGAGTYYPDSVKG VH 50-66 ofSEQ ID NO: 198 E26.2 CDR-H3 Residues GGVTKGYFDV VH 99-108 of SEQ IDNO: 198 199 E26.2 Same as Parental VL DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQIT E26.2 CDR-L1 Residues RASGNIHNYLT VL 24-34 of SEQ IDNO: 199 E26.2 CDR-L2 Residues NAKTLAD VL 50-56 of SEQ ID NO: 199 E26.2CDR-L3 Residues QHFWSIPYT VL 89-97 of SEQ ID NO: 199 200 E26.11EVQLVESGGGVVQPGRSLRLSCSASGFIFS VH RYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPED TAVYYCARGGVYKGYFDVWGQGTPVTVSS E26.11CDR-H1 Residues RYDMS VH 31-35 of SEQ ID NO: 200 E26.11 CDR-H2 ResiduesYISHGGAGTYYPDSVKG VH 50-66 of SEQ ID NO: 200 E26.11 CDR-H3 ResiduesGGVYKGYFDV VH 99-108 of SEQ ID NO: 200 201 E26.11DIQMTQSPSSLSASVGDRVTITCRASGNIH VL NYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH FWSIPYTFGQGTKLQIT E26.11 CDR-L1 ResiduesRASGNIHNYLT VL 24-34 of SEQ ID NO: 201 E26.11 CDR-L2 Residues NAKTLAD VL50-56 of SEQ ID NO: 201   E26.11 CDR-L3 Residues QHFWSIPYT VL 89-97 ofSEQ ID NO: 201 202 E26.12 EVQLVESGGGVVQPGRSLRLSCAASGFIFS VHRYDMSWVRQAPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS E26.12 CDR-H1 Residues  RYDMS VH 31-35 ofSEQ ID NO: 202   E26.12 CDR-H2 Residues YISHGGAGTYYPDSVKG VH 50-66 ofSEQ ID NO: 202   E26.12 CDR-H3 Residues GGVTKGYFDV VH 99-108 of SEQ IDNO: 202 203 E26.12 Same as Parental VL DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQIT E26.12 CDR-L1 Residues RASGNIHNYLT VL 24-34 of SEQ IDNO: 203 E26.12 CDR-L2 Residues NAKTLAD VL 50-56 of SEQ ID NO: 203 E26.12CDR-L3 Residues QHFWSIPYT VL 89-97 of SEQ ID NO: 203 204 E26.13EVQLVESGGGVVQPGRSLRLSCSASGFIFS VH RYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPED TGVYFCARGGVTKGYFDVWGQGTPVTVSS E26.13CDR-H1 Residues RYDMS VH 31-35 of SEQ ID NO: 204 E26.13 CDR-H2 ResiduesYISHGGAGTYYPDSVKG VH 50-66 of SEQ ID NO: 204 E26.13 CDR-H3 ResiduesGGVTKGYFDV VH 99-108 of SEQ ID NO: 204 205 E26.13 Same as Parental VLDIQMTQSPSSLSASVGDRVTITCRASGNIH NYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH FWSIPYTFGQGTKLQIT E26.13 CDR-L1 ResiduesRASGNIHNYLT VL 24-34 of SEQ ID NO: 205 E26.13 CDR-L2 Residues NAKTLAD VL50-56 of SEQ ID NO: 205 E26.13 CDR-L3 Residues QHFWSIPYT VL 89-97 ofSEQ ID NO: 205 206 E26.13 EVQLVESGGGVVQPGRSLRLSCSASGFIFS JM VHRYDMSWVRQAPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTTVTVSS E26.13 CDR-H1 Residues RYDMS JM VH31-35 of SEQ ID NO: 206 E26.13 CDR-H2 Residues YISHGGAGTYYPDSVKG JM VH50-66 of SEQ ID NO: 206 E26.13 CDR-H3 Residues GGVTKGYFDV JM VH99-108 of SEQ ID NO: 206 207 E26.13 DIQMTQSPSSLSASVGDRVTITCRASGNIH JM VLNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLEIKR E26.13 CDR-L1 Residues RASGNIHNYLT JM VL 24-34 ofSEQ ID NO: 207 E26.13 CDR-L2 Residues NAKTLAD JM VL 50-56 of SEQ IDNO: 207 E26.13 CDR-L3 Residues QHFWSIPYT JM VL 89-97 of SEQ ID NO: 207208 E26.35 EVQLVESGGGVVQPGRSLRLSCSASGFIFS VHRYDMSWVRQAPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLFLQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSS E26.35 CDR-H1 Residues RYDMS VH 31-35 ofSEQ ID NO: 208 E26.35 CDR-H2 Residues YISHGGAGTYYPDSVKG VH 50-66 ofSEQ ID NO: 208 E26.35 CDR-H3 Residues GGVYKGYFDV VH 99-108 of SEQ IDNO: 208 209 E26.35 DIQMTQSPSSLSASVGDRVTITCRASGNIH VLNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQIT E26.35 CDR-L1 Residues RASGNIHNYLT VL 24-34 of SEQ IDNO: 209 E26.35  CDR-L2 Residues NAKTLAD VL 50-56 of SEQ ID NO: 209E26.35 CDR-L3 Residues QHFWSIPYT VL 89-97 of SEQ ID NO: 209 210 E26.37EVQLVESGGGVVQPGRSLRLSCSASGFIFS VH KYDMSWVRQAPGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDSLRPED TGVYYCARGGVYKGYFDVWGQGTPVTVSS E26.37CDR-H1 Residues KYDMS VH 31-35 of SEQ ID NO: 210 E26.37 CDR-H2 ResiduesYISHGGAGTYYPDSVKG VH 50-66 of SEQ ID NO: 210 E26.37 CDR-H3 ResiduesGGVYKGYFDV VH 99-108 of SEQ ID NO: 210 211 E26.37DIQMTQSPSSLSASVGDRVTITCRASGNIY VL TYLTWYQQTPGKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQH FWTLPYTFGQGTKLQIT E26.37 CDR-L1 ResiduesRASGNIYTYLT VL 24-34 of SEQ ID NO: 211 E26.37 CDR-L2 Residues NAKTLAD VL50-56 of SEQ ID NO: 211 E26.37 CDR-L3 Residues QHFWTLPYT VL 89-97 ofSEQ ID NO: 211

Example 2 Functional Characterization of IL-1β Antibodies Example 2.1IL-1β Enzyme-Linked Immunosorbent Assay Protocol

To determine if the anti-IL-1β mAbs bind to human IL-1β, ELISA plates(Nunc, MaxiSorp, Rochester, N.Y.) were incubated overnight at 4° C. withanti-human Fc antibody diluted in Pierce Coat buffer at 2 μg/ml (JacksonImmunoresearch, West Grove, Pa.). Plates were washed five times inwashing buffer (PBS containing 0.05% Tween 20), and blocked for 1 hourat 25° C. with 200 μl per well superblock blocking buffer (Thermoscientific, No. 37515). Blocking buffer was removed by tapping plates,and 2 μg/ml of each antibody in PBS containing 10% Superblock, 0.5%Tween-20 was added to the wells at 100 μl/well and incubated at 25° C.for 1 hour. The wells were washed five times in 1XPBST, and 1 μg/mlbiotinylated antigen was titrated at 1:6 serial dilutions (for a rangeof μg to pg in PBS containing 10% Superblock, 0.05% Tween 20). Eachdilution of antigen was then added to the plates and incubated for 1hour at 25° C. The wells were washed five times in 1×PBST and incubatedfor 1 hour at 25° C. with polyHRP streptavidin (KPL No. 474-3000,Gaithersburg, Md.). The wells were washed five times in 1×PBST, and 100μl of ULTRA-TMB ELISA (Pierce, Rockford, Ill.) were added per well.Following color development the reaction was stopped with 1N HCL andabsorbance at 450 nM was measured. The results are shown in Table 11,and the numerical value indicates binding of anti-IL-1β antibodies tohuman IL-1β.

TABLE 11 Binding of Antibodies to Human IL-1β by ELISA MAb EC50 inhIL-1β ELISA (pM) E26.1 12.9 E26.2 567 E26.11 14 E26.12 306 E26.13 7.2E26.13 JM 7.4 E26.35 10.4 E26.37 17.7

Example 2.2 Neutralizing Potency of IL-1β Antibodies

To examine the functional activity of the anti-human IL-1β antibodies inthe invention, the antibodies were used in an MRC-5 assay that measuresthe ability of the antibody to inhibit IL-1β activity. The MRC-5 cellline is a human lung fibroblast cell line that produces IL-8 in responseto human IL-1β in a dose-dependent manner. This cell line also producesIL-8 in response to cynomolgus IL-1β (cyno IL-1β) MRC-5 cells wereoriginally obtained from ATCC and subcultured in 10% FBS complete MEMand grown at 37° C. in a 5% CO₂ incubator. To determine an antibody'sneutralizing potency against IL-1β, antibodies (50 μl) were added to a96 well plate (1E-7 to 1E-15 M final concentration range) andpre-incubated with 50 μl of human or cyno IL-1β (50 μg/mL finalconcentration) for 1 hour at 37° C., 5% CO₂. Antigen antibody complexes(100 μL) were then added to MRC-5 cells (plated 24 hour previously at aconcentration of 1E5/ml at 100 μl cells/well). Assay plates wereincubated overnight at 37° C. in a 5% CO₂ incubator. Antibody potencywas determined by its ability to inhibit IL-8 production. Human IL-8production was measured by a chemiluminescence-based assay. Table 12summarizes antibody potencies to human and cyno IL-1β.

TABLE 12 Neutralizing Potency of IL-1β Antibodies Potency (pM)Anti-IL-1β mAb hIL-1β (pM) Cyno IL-1β (pM) E26.1 9.7 ND E26.13 15.7 8.4E26.35 7.2 3.0 E26.37 2.2 ND ND: Not determined.

Example 2.3 Affinity Measurement of IL-1β Antibodies by Surface PlasmonResonance

The BIACORE assay (Biacore, Inc, Piscataway, N.J.) determines theaffinity of antibodies with kinetic measurements of on-rate and off-rateconstants. Binding of antibodies to recombinant purified human IL-1β andcynomolgus IL-1β was determined by surface plasmon resonance-basedmeasurements with a Biacore® 3000 instrument (Biacore® AB, Uppsala,Sweden) using running HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mMEDTA, and 0.005% surfactant P20) at 25° C. All chemicals were obtainedfrom Biacore® AB (Uppsala, Sweden) or otherwise from a different sourceas described herein. Approximately 5000 RU of goat anti-mouse IgG,(Fcγ), fragment specific polyclonal antibody (Pierce Biotechnology Inc,Rockford, Ill.) diluted in 10 mM sodium acetate (pH 4.5) was directlyimmobilized across a CM5 research grade biosensor chip using a standardamine coupling kit according to manufacturer's instructions andprocedures at 25 μg/ml. Unreacted moieties on the biosensor surface wereblocked with ethanolamine. Modified carboxymethyl dextran surface inflowcell 2 and 4 was used as a reaction surface. Unmodifiedcarboxymethyl dextran without goat anti-mouse IgG in flow cell 1 and 3was used as the reference surface. For kinetic analysis, rate equationsderived from the 1:1 Langmuir binding model were fitted simultaneouslyto association and dissociation phases of all eight injections (usingglobal fit analysis) with the use of Biaevaluation 4.0.1 software.Purified antibodies were diluted in HEPES-buffered saline for captureacross goat anti-human IgG specific reaction surfaces. Antibodies to becaptured as a ligand (25 μg/ml) were injected over reaction matrices ata flow rate of 5 μl/minute. The association and dissociation rateconstants, k_(on) (unit M⁻¹ s⁻¹) and k_(off) (unit s⁻¹) were determinedunder a continuous flow rate of 25 μl/min. Rate constants were derivedby making kinetic binding measurements at ten different antigenconcentrations ranging from 10-200 nM. The equilibrium dissociationconstant (unit M) of the reaction between antibodies and the targetantigen was then calculated from the kinetic rate constants by thefollowing formula: K_(D)=k_(off)/k_(on). Binding is recorded as afunction of time and kinetic rate constants are calculated. In thisassay, on-rates as fast as 10⁶ M⁻¹ s⁻¹ and off-rates as slow as 10⁻⁶ s⁻¹can be measured. Table 13 shows the affinity measurements for humananti-IL-1β antibodies.

TABLE 13 Affinity of Antibodies to Human and Cyno IL-1β by Biacore HumanCyno IL-1β IL-1β E26.2 (M) 5.28 × 10⁻¹¹ ND Kon (1/Ms) 8.95 × 10⁵ ND Koff(1/s) 4.72 × 10⁻⁵ ND E26.12 (M) 7.86 × 10⁻¹¹ ND Kon (1/Ms) 9.33 × 10⁵ NDKoff (1/s) 7.37 × 10⁻⁵ ND E26.13 (M) 4.45 × 10⁻¹¹ 1.46 × 10⁻¹¹ Kon(1/Ms) 9.5 × 10⁵ 1.23 × 10⁶ Koff (1/s) 4.23 × 10⁻⁵ 1.79 × 10⁻⁵ E26.35(M) 2.39 × 10⁻¹¹ 1.4 × 10⁻¹¹ Kon (1/Ms) 1.02 × 10⁶ 9.21 × 10⁵ Koff (1/s)2.50 × 10⁻⁵ 1.29 × 10⁻⁵ ND: Not determined.

Example 3 Generation of IL-1α/β DVD-Ig™ Molecules Example 3.1Construction of IL-1α/β DVD-Ig DNA Constructs

An anti-IL-1α antibody (“X3”; see, PCT Publication No. WO 95/14780)variable domain was combined with multiple IL-1β antibody variabledomains into a DVD-Ig format (Wu et al., Nature Biotechnol., 25:1290-1297 (2007); PCT Publication No. WO 2007/024715 A2) by overlappingPCR amplification with intervening linker DNA sequences. X3 was alsomutated in the J-region of the variable heavy and light region, calledX3 JM VH and X₃ JM VL, respectively, to remove non-germline frameworkmutations. The amplified PCR products were subcloned into expressionvectors suitable for transient expression in HEK293 cells and the openreading frame regions were confirmed by sequencing before DVD-Igexpression.

Example 3.2 Expression and Production of IL-1α/β DVD-Ig Binding Proteins

After DNA sequence confirmation, all DVD-Ig DNA constructs were expandedin E. coli and DNAs were purified using Qiagen Hispeed Maxi Prep(Catalog No. 12662, QIAGEN). DVD-Ig DNA was transfected into log phase293E cells (0.5×10⁶/ml, viability >95%) by mixing PEI and DNA at a 2:1ratio with 0.2 μg/ml heavy chain DNA and 0.3 μg/ml light chain DNA.DNA:PEI complex was formed at room temperature in TC hood for fifteenminutes before adding to 293E cells. Twenty four later, 0.5% TN1 wasadded to 293E cells. At day five, supernatant was collected for humanIgG1 titer measurement. Cell supernatant was harvested at day seven andfiltered through a 0.2 μM PES filter. Supernatant was purified by usingProtein A Sepharose Affinity Chromatography according to manufacturer'sinstruction. Purified DVD-Igs were eluted off the column by 0.1 Mglycine (pH 2.99) and dialyzed into 15 mM histidine buffer (pH 6.0)immediately. The binding proteins were quantitated by A280 and analyzedby mass spectrometry and SEC.

Example 3.3 Sequences of IL-1α/β DVD-Ig Constructs

Amino acid sequences of heavy and light chains of DVD-Ig proteinscapable of binding human IL-1β and human IL-1α were determined. Theamino acid sequences of variable heavy chain (VH), variable light chain(VL), constant light chain (CL), and constant heavy chain (CH) regionsof IL-1α/β DVD-Ig binding proteins are shown in Table 14, below. InTable 14, the amino acid sequences for the E26.13 and E26.35 VL regionsare designated SEQ ID NO:238 and SEQ ID NO:239, respectively, instead ofSEQ ID NO:205 and SEQ ID NO:209 as previously shown in Table 10, toaccount for the inclusion of a C-terminal arginine (R) residue. ThisC-terminal arginine residue is understood by those skilled in the art ofantibody engineering to be the amino acid residue at the junction of VLand CL kappa regions in an IgG molecule and is sometimes included in theCL region or, as in Table 14 below, the VL region.

TABLE 14 Sequences of Variable and Constant Regions of IL-1α/βDVD-Ig Binding Proteins Protein Sequence Protein regionSequence Identifier 12345678901234567890 E26.13-SS-X3 SEQ ID NO: 212EVQLVESGGGVVQPGRSLRL DVD-Ig HEAVY SCSASGFIFSRYDMSWVRQA VARIABLEPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQG TLVTFSS E26.13 VH SEQ ID NO: 204EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTPVTVSS LINKERSEQ ID NO: 33 ASTKGP X3 VH SEQ ID NO: 213 QVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SS CH SEQ ID NO: 214ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK E26.13-SS-X3 SEQ ID NO: 215DIQMTQSPSSLSASVGDRVT DVD-Ig LIGHT ITCRASGNIHNYLTWYQQTP VARIABLEGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQGTKLQITRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEHK RE26.13 VL SEQ ID NO: 238 DIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITRLINKER SEQ ID NO: 35  TVAAP X3 VL SEQ ID NO: 216 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGG GTKVEHKR CL SEQ ID NO: 5 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC E26.13-LL-X3 SEQ ID NO: 217EVQLVESGGGVVQPGRSLRL DVD-Ig HEAVY SCSASGFIFSRYDMSWVRQA VARIABLEPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSSA STKGPSVFPLAPQVQLVESG GGVVQPGRSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWV AAVSYDGSNKYYAESVKGRF TISRDNSKNILFLQMDSLRLEDTAVYYCARGRPKVVIPAP LAHWGQGTLVTFSS E26.13 VH SEQ ID NO: 204EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTPVTVSS LINKERSEQ ID NO: 34 ASTKGPSVFPLAP X3 VH SEQ ID NO: 213 QVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SS CH SEQ ID NO: 214ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK E26.13-LL-X3 SEQ ID NO: 218DIQMTQSPSSLSASVGDRVT DVD-Ig LIGHT ITCRASGNIHNYLTWYQQTP VARIABLEGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQGTKLQITRTVAAPSVFIFPP DIQMTQSPSSVSASVGDRVT ITCRASQGISSWLAWYQQKPGKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKRE26.13 VL SEQ ID NO: 238 DIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITRLINKER SEQ ID NO: 36 TVAAPSVFIFPP X3 VL SEQ ID NO: 216DIQMTQSPSSVSASVGDRVT ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKR CL SEQ ID NO: 5TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC X3-SS-E26.13SEQ ID NO: 219 QVQLVESGGGVVQPGRSLRL DVD-Ig HEAVY SCTASGFTFSMFGVHWVRQAVARIABLE PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTF SSASTKGPEVQLVESGGGVV QPGRSLRLSCSASGFIFSRYDMSWVRQAPGKGLEWVAYIS HGGAGTYYPDSVKGRFTISR DNSKNTLFLQMDSLRPEDTGVYFCARGGVTKGYFDVWGQG TPVTVSS X3 VH SEQ ID NO: 213 QVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SS LINKER SEQ ID NO: 33 ASTKGPE26.13 VH SEQ ID NO: 204 EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS CH SEQ ID NO: 214 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK X3-SS-E26.13 SEQ ID NO: 220 DIQMTQSPSSVSASVGDRVT DVD-Ig LIGHTITCRASQGISSWLAWYQQKP VARIABLE GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGG GTKVEHKRTVAAPDIQMTQS PSSLSASVGDRVTITCRASGNIHNYLTWYQQTPGKAPKLL IYNAKTLADGVPSRFSGSGS GTDYTFTISSLQPEDIATYYCQHFWSIPYTFGQGTKLQIT R X3 VL SEQ ID NO: 216 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGG GTKVEHKR LINKER SEQ ID NO: 35 TVAAP E26.13 VLSEQ ID NO: 238 DIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITRCL SEQ ID NO: 5 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGECX3-LL-E26.13 SEQ ID NO: 221 QVQLVESGGGVVQPGRSLRL DVD-Ig HEAVYSCTASGFTFSMFGVHWVRQA VARIABLE PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SSASTKGPSVFPLAPEVQLVESGGGVVQPGRSLRLSCSAS GFIFSRYDMSWVRQAPGKGL EWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLFLQMDS LRPEDTGVYFCARGGVTKGY FDVWGQGTPVTVSS X3 VHSEQ ID NO: 213 QVQLVESGGGVVQPGRSLRL SCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTF SS LINKER SEQ ID NO: 34 ASTKGPSVFPLAP E26.13 VHSEQ ID NO: 204 EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSS CH SEQ ID NO: 214 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK X3-LL- E26.13 SEQ ID NO: 222 DIQMTQSPSSVSASVGDRVTDVD-Ig LIGHT ITCRASQGISSWLAWYQQKP VARIABLE GKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKRTVAAPSVFIFPPDIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITR X3 VL SEQ ID NO: 216DIQMTQSPSSVSASVGDRVT ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKR LINKER SEQ ID NO: 36TVAAPSVFIFPP E26.13 VL SEQ ID NO: 238 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWSIPYTFGQ GTKLQITR CL SEQ ID NO: 5 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC E26.35-SS-X3 JM SEQ ID NO: 226EVQLVESGGGVVQPGRSLRL DVD-Ig HEAVY SCSASGFIFSRYDMSWVRQA VARIABLEPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQG TLVTVSS E26.35 VH SEQ ID NO: 208EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLF LQMDSLRAEDTAVYYCARGG VYKGYFDVWGQGTPVTVSS LINKERSEQ ID NO: 33 ASTKGP X3 JM VH SEQ ID NO: 227 QVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTV SS CH SEQ ID NO: 214ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK E26.35-SS-X3 JM SEQ ID NO: 228DIQMTQSPSSLSASVGDRVT DVD-Ig LIGHT ITCRASGNIHNYLTWYQQTP VARIABLEGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQGTKLQITRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEIK RE26.35 VL SEQ ID NO: 239 DIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITRLINKER SEQ ID NO: 35 TVAAP X3 JM VL SEQ ID NO: 229 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGG GTKVEIKR CL SEQ ID NO: 5 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC E26.13 JM-SS-X3 SEQ ID NO: 230EVQLVESGGGVVQPGRSLRL DVD-Ig HEAVY SCSASGFIFSRYDMSWVRQA VARIABLEPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTTVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQG TLVTFSS E26.13 JM VH SEQ ID NO: 206EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTTVTVSS LINKERSEQ ID NO: 33 ASTKGP X3 VH SEQ ID NO: 213 QVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SS CH SEQ ID NO: 214ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK E26.13 JM-SS-X3 SEQ ID NO: 231DIQMTQSPSSLSASVGDRVT DVD-Ig LIGHT ITCRASGNIHNYLTWYQQTP VARIABLEGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQGTKLEIKRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEHK RE26.13 JM VL SEQ ID NO: 207 DIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLEIKRLINKER SEQ ID NO: 35 TVAAP X3 VL SEQ ID NO: 216 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGG GTKVEHKR CL SEQ ID NO: 5 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC E26.35-SS-X3 SEQ ID NO: 232EVQLVESGGGVVQPGRSLRL DVD-Ig HEAVY SCSASGFIFSRYDMSWVRQA VARIABLEPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRAEDTAVYYCARGGVYKGYFDVWGQGTPVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQG TLVTFSS E26.35 VH SEQ ID NO: 208EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLF LQMDSLRAEDTAVYYCARGG VYKGYFDVWGQGTPVTVSS LINKERSEQ ID NO: 33 ASTKGP X3 VH SEQ ID NO: 213 QVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTF SS CH SEQ ID NO: 214ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK E26.35-SS-X3 SEQ ID NO: 233DIQMTQSPSSLSASVGDRVT DVD-Ig LIGHT ITCRASGNIHNYLTWYQQTP VARIABLEGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQGTKLQITRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQ GISSWLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYY CQQTSSFLLSFGGGTKVEHK RE26.35 VL SEQ ID NO: 239 DIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLQITRLINKER SEQ ID NO: 35 TVAAP X3 VL SEQ ID NO: 216 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGG GTKVEHKR CL SEQ ID NO: 5 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC E26.13-SS-X3 JM SEQ ID NO: 234EVQLVESGGGVVQPGRSLRL DVD-Ig HEAVY SCSASGFIFSRYDMSWVRQA VARIABLEPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTPVTVSSA STKGPQVQLVESGGGVVQPG RSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDG SNKYYAESVKGRFTISRDNS KNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQG TLVTVSS E26.13 VH SEQ ID NO: 204EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQA PGKGLEWVAYISHGGAGTYYPDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTPVTVSS LINKERSEQ ID NO: 33 ASTKGP X3 JM VH SEQ ID NO: 227 QVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQA PGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGR PKVVIPAPLAHWGQGTLVTV SS CH SEQ ID NO: 214ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK E26.13-SS-X3 JM SEQ ID NO: 235DIQMTQSPSSLSASVGDRVT Anti-IL-1alpha/beta ITCRASGNIHNYLTWYQQTPDVD-Ig LIGHT GKAPKLLIYNAKTLADGVPS VARIABLE RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWSIPYTFGQ GTKLQITRTVAAPDIQMTQS PSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLL IYEASNLETGVPSRFSGSGS GSDFTLTISSLQPEDFATYYCQQTSSFLLSFGGGTKVEIK R E26.13 VL SEQ ID NO: 238 DIQMTQSPSSLSASVGDRVTITCRASGNIHNYLTWYQQTP GKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQPEDIATYYCQHFWSIPYTFGQ GTKLQITR LINKER SEQ ID NO: 35 TVAAP X3 JM VLSEQ ID NO: 229 DIQMTQSPSSVSASVGDRVT ITCRASQGISSWLAWYQQKPGKAPKLLIYEASNLETGVPS RFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEIKRCL SEQ ID NO: 5 TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGECE26.13 JM-LL-X3 SEQ ID NO: 236 EVQLVESGGGVVQPGRSLRL DVD-Ig HEAVYSCSASGFIFSRYDMSWVRQA VARIABLE PGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLFLQMDSLRPEDTGVYFCARGG VTKGYFDVWGQGTTVTVSSA STKGPSVFPLAPQVQLVESGGGVVQPGRSLRLSCTASGFT FSMFGVHWVRQAPGKGLEWV AAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMDSLRL EDTAVYYCARGRPKVVIPAP LAHWGQGTLVTFSS E26.13 JM VHSEQ ID NO: 206 EVQLVESGGGVVQPGRSLRL SCSASGFIFSRYDMSWVRQAPGKGLEWVAYISHGGAGTYY PDSVKGRFTISRDNSKNTLF LQMDSLRPEDTGVYFCARGGVTKGYFDVWGQGTTVTVSS LINKER SEQ ID NO: 34 ASTKGPSVFPLAP X3 VHSEQ ID NO: 213 QVQLVESGGGVVQPGRSLRL SCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDGSNKYY AESVKGRFTISRDNSKNILF LQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTF SS CH SEQ ID NO: 214 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK E26.13 JM-LL-X3 SEQ ID NO: 237 DIQMTQSPSSLSASVGDRVTDVD-Ig LIGHT ITCRASGNIHNYLTWYQQTP VARIABLE GKAPKLLIYNAKTLADGVPSRFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLEIKRTVAAPSVFIFPPDIQMTQSPSSVSASVGDRVT ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKR E26.13 JM VLSEQ ID NO: 207 DIQMTQSPSSLSASVGDRVT ITCRASGNIHNYLTWYQQTPGKAPKLLIYNAKTLADGVPS RFSGSGSGTDYTFTISSLQP EDIATYYCQHFWSIPYTFGQ GTKLEIKRLINKER SEQ ID NO: 36 TVAAPSVFIFPP X3 VL SEQ ID NO: 216DIQMTQSPSSVSASVGDRVT ITCRASQGISSWLAWYQQKP GKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQP EDFATYYCQQTSSFLLSFGG GTKVEHKR CL SEQ ID NO: 5TVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK HKVYACEVTHQGLSSPVTKS FNRGEC Linker sequences areindicated as underlined residues.

Example 4 Functional Characterization of IL-1α/β DVD-Ig Proteins Example4.1 IL-1α/β Enzyme-Linked Immunosorbent Assay Protocol

IL-1β and IL-1α binding by IL-1β/α DVD-Ig binding proteins was assessedby ELISA (assay described above, Example 2.1). Results are shown inTable 15.

TABLE 15 BINDING of IL-1α/β DVD-Ig Proteins to Human IL-1α or IL-1β byELISA EC50 in hIL-1β EC50 in hIL-1α MAb ELISA (pM) ELISA (pM)E26.13-LL-X3 8.1 5.8 E26.13-SS-X3 7.5 6.4 E26.13-SS-X3 JM 6.9 4.3E26.35-SS-X3 8 6.2 E26.35-SS-X3 JM 6.3 4.0 X3-SS-E26.13 70 4.5

Example 4.2 IL-1α/β Bioassay and Neutralization Assay

MRCS cells were plated at 1.5−2×10⁴ cells per well in a 100 μL volumeand incubated overnight at 37° C., 5% CO₂. A 20 μg/mL working stock ofDVD-Ig (4× concentrated) was prepared in complete MEM medium. An eightpoint serial dilution was performed (5 μg/mL-0.0003 μg/mL) in completeMEM in Marsh dilution plates. Sixty-five μL/well of each antibodydilution was added in quadruplicate to a 96 well v-bottom (Costar No.3894) plate and 65 μL of a 200 pg/mL solution of IL-1α or IL-1β or 65 μLof a mixed solution containing a 50 pg/mL solution of both IL-1α andIL-1β. Control wells received 65 μL 200 pg/ml of IL-1α or IL-1β or 50pg/mL mixed IL-1α/β (4× concentrated) plus 65 μL MEM media and mediacontrol wells received 130 μL of media. Following a 1 hour incubation,100 μL of the DVD-Ig/Ag mixture was added to the MRC5 cells. All wellvolumes were equal to 200 μL. All plate reagents were then 1×concentrated. After a 16-20 hour incubation, the well contents (150 μL)were transferred into a 96-well round bottom plate (Costar No. 3799) andplaced in a −20° C. freezer. The supernatants were tested for hIL-8levels by using a human IL-8 ELISA kit (R&D Systems, Minneapolis, Minn.)or MSD hIL-8 (chemiluminescence kit). Neutralization potency wasdetermined by calculating percent inhibition relative to the IL-1α,IL-1β, or the IL-1α/β alone control value (Table 16).

TABLE 16 Potency of IL-1α/β DVD-Ig Molecules on Human IL-1α and IL-1βand Cynomolgus IL-1α and IL-1β Potency (pM) IL-1α/β DVD-Ig hIL-1β hIL-1αCyno IL-1β Cyno IL-1α E26.13-LL-X3 18.3 10.2 16.7 1053 E26.13-SS-X3 16.016.2 8.4 955 E26.13-SS-X3 JM 28.3 26.5 17.6 1880 E26.35-SS-X3 1.8 25.80.6 474 X3-LL-E26.13 1470 8.9 ND ND X3-SS-E26.13 2676 7.6 ND ND ND: Notdetermined.

Example 4.3 Affinity Measurement of IL-1α/β DVD-Ig Molecules

The binding of IL-1α/β DVD-Igs to purified recombinant human IL-1β andIL-1α and cynomolgus IL-1β and IL-1α were determined using surfaceplasmon resonance as described in Example 2.3 and results are shown inTable 17.

TABLE 17 Affinity Measurement of IL-1α/β DVD-Ig Molecules Human HumanCyno Cyno IL-1β IL-1α IL-1β IL-1α E26.13-LL-X3 7.82 × 10⁻¹² 6.15 × 10⁻¹²1.24 × 10⁻¹¹ 3.24 × 10⁻⁹ Kon (1/Ms) 1.45 × 10⁶ 6.46 × 10⁵ 1.74 × 10⁶3.15 × 10⁵ Koff (1/s) 1.13 × 10⁻⁵ 3.9 × 10⁻⁶ 2.16 × 10⁻⁵ 1.05 × 10⁻³E26.13-SS-X3 2.06 × 10⁻¹¹ 7.61 × 10⁻¹² 1.53 × 10⁻¹¹ 4.11 × 10⁻⁹ Kon(1/Ms) 1.77 × 10⁶ 1.98 × 10⁵ 1.45 × 10⁶ 6.37 × 10⁴ Koff (1/s) 3.61 ×10⁻⁵ 1.5 × 10⁻⁶ 2.22 × 10⁻⁵ 2.61 × 10⁻⁴ E26.35-SS-X3 5.03 × 10⁻¹² 1.33 ×10⁻¹¹ 1.06 × 10⁻¹¹ 3.27 × 10⁻⁹ Kon (1/Ms) 1.32 × 10⁶ 1.62 × 10⁵ 1.84 ×10⁶ 7.24 × 10⁴ Koff (1/s) 6.81 × 10⁻⁶ 2.14 × 10⁻⁶ 1.94 × 10⁻⁵ 2.36 ×10⁻⁴

INCORPORATION BY REFERENCE

The present invention incorporates by reference in their entiretytechniques well known in the field of molecular biology and drugdelivery. These techniques include, but are not limited to, techniquesdescribed in the following publications: Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Ausubel,F. M. et al. eds., Short Protocols In Molecular Biology (4th Ed. 1999)John Wiley & Sons, NY. (ISBN 0-471-32938-X). Controlled DrugBioavailability Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Giegé et al., Chapter 1, InCrystallization of Nucleic Acids and Proteins, A Practical Approach, 2nded., (Ducruix and Giegé, eds.) (Oxford University Press, New York, 1999)pp. 1-16; Goodson, J. M., Chapter 6, In Medical Applications ofControlled Release, Vol. II, Applications and Evaluation, (Langer andWise, eds.) (CRC Press, Inc., Boca Raton, 1984), pp. 115-138; Hammerlinget al., eds., “Monoclonal Antibodies and T-Cell Hybridomas,” In ResearchMonographs in Immunology, vol. 3 (J. L. Turk, General Editor) (Elsevier,New York, 1981), pp. 563-587; Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Kabat etal., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md. (1987); Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Kontermann and Diibel, eds., Antibody Engineering (2001)Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5); Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990);Lu and Weiner eds., Cloning and Expression Vectors for Gene FunctionAnalysis (2001) BioTechniques Press. Westborough, Mass. 298 pp.(ISBN¹-881299-21-X); Goodson, J. M., Medical Applications of ControlledRelease, (Langer and Wise, eds.) (CRC Press, Boca Raton, 1974); Old andPrimrose, Principles of Gene Manipulation: An Introduction To GeneticEngineering (3d Ed. 1985) Blackwell Scientific Publications, Boston;Studies in Microbiology, V. 2:409 pp. (ISBN 0-632-01318-4); Sambrook, J.et al., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold SpringHarbor Laboratory Press, NY. Vols. 1-3 (ISBN 0-87969-309-6); Sustainedand Controlled Release Drug Delivery Systems, (J. R. Robinson, ed.)(Marcel Dekker, Inc., New York, 1978); Winnacker, E. L. From Genes ToClones: Introduction To Gene Technology (1987) VCH Publishers, N.Y.(translated by Horst Ibelgaufts), 634 pp. (ISBN 0-89573-614-4).

The contents of all cited references (including literature references,patents, patent applications, and websites) that are cited throughoutthis application are hereby expressly incorporated by reference in theirentirety, as are the references cited therein. The practice of thepresent invention will employ, unless otherwise indicated, conventionaltechniques of immunology, molecular biology and cell biology, which arewell known in the art.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. The scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

1-47. (canceled)
 48. A method for treating a subject for a disorder inwhich IL-1 activity is detrimental comprising administering to thesubject a binding protein or a crystal thereof, wherein the bindingprotein comprises first and second polypeptide chains, wherein saidfirst polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n,wherein: VD1 is a first heavy chain variable domain; VD2 is a secondheavy chain variable domain; C is a heavy chain constant domain; X1 is alinker with the proviso that it is not CH1; X2 is an Fc region; and n isindependently 0 or 1; and wherein said second polypeptide chaincomprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first lightchain variable domain; VD2 is a second light chain variable domain; C isa light chain constant domain; X1 is a linker with the proviso that itis not CH1; X2 does not comprise an Fc region; and n is independently 0or 1; wherein, in said first polypeptide chain, VD1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 60-148,196, 198, 200, 202, 204, 206, 208 and 210; and VD2 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 213 and227; wherein, in said second polypeptide chain, VD1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 149-189,197, 199, 201, 203, 205, 207, 209 and 211; and VD2 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 216 and229; and wherein the binding protein binds human IL-1β and human IL-1α,such that treatment for the disorder is achieved.
 49. The method ofclaim 48, wherein said disorder is selected from the group consistingof: diabetes; uveitis; neuropathic pain; osteoarthritic pain;inflammatory pain; rheumatoid arthritis; osteoarthritis; juvenilechronic arthritis; septic arthritis; Lyme arthritis; psoriaticarthritis; reactive arthritis; spondyloarthropathy; systemic lupuserythematosus (SLE); Crohn's disease; ulcerative colitis; inflammatorybowel disease; autoimmune diabetes; insulin dependent diabetes mellitus;thyroiditis; asthma; allergic diseases; psoriasis; dermatitis;scleroderma; graft versus host disease; organ transplant rejection;acute immune disease associated with organ transplantation; chronicimmune disease associated with organ transplantation; sarcoidosis;atherosclerosis; disseminated intravascular coagulation (DIC);Kawasaki's disease; Grave's disease; nephrotic syndrome; chronic fatiguesyndrome; Wegener's granulomatosis; Henoch-Schoenlein purpurea;microscopic vasculitis of the kidneys; chronic active hepatitis;autoimmune uveitis; septic shock; toxic shock syndrome; sepsis syndrome;cachexia; infectious diseases; parasitic diseases; acute transversemyelitis; Huntington's chorea; Parkinson's disease; Alzheimer's disease;stroke; primary biliary cirrhosis; hemolytic anemia; malignancies; heartfailure; myocardial infarction; Addison's disease; sporadicpolyglandular deficiency type I; polyglandular deficiency type II(Schmidt's syndrome); acute respiratory distress syndrome (ARDS);alopecia; alopecia greata; seronegative arthropathy; arthropathy;Reiter's disease; psoriatic arthropathy; ulcerative colitic arthropathy;enteropathic synovitis; chlamydia; Yersinia and Salmonella associatedarthropathy; spondyloarthropathy; atheromatous disease/arteriosclerosis;atopic allergy; autoimmune bullous disease; pemphigus vulgaris;pemphigus foliaceus; pemphigoid; linear IgA disease; autoimmunehaemolytic anaemia; Coombs positive haemolytic anaemia; acquiredpernicious anaemia; juvenile pernicious anaemia; myalgicencephalitis/Royal Free disease; chronic mucocutaneous candidiasis;giant cell arteritis (GCA); primary sclerosing hepatitis; cryptogenicautoimmune hepatitis; acquired immunodeficiency syndrome (AIDS);acquired immunodeficiency related diseases; hepatitis B; hepatitis C;common varied immunodeficiency (common variable hypogammaglobulinaemia);dilated cardiomyopathy; female infertility; ovarian failure; prematureovarian failure; fibrotic lung disease; cryptogenic fibrosingalveolitis; post-inflammatory interstitial lung disease; interstitialpneumonitis; connective tissue disease associated interstitial lungdisease; mixed connective tissue disease associated lung disease;systemic sclerosis associated interstitial lung disease; rheumatoidarthritis associated interstitial lung disease; systemic lupuserythematosus associated lung disease; dermatomyositis/polymyositisassociated lung disease; Sjögren's disease associated lung disease;ankylosing spondylitis associated lung disease; vasculitic diffuse lungdisease; haemosiderosis associated lung disease; drug-inducedinterstitial lung disease; fibrosis; radiation fibrosis; bronchiolitisobliterans; chronic eosinophilic pneumonia; lymphocytic infiltrativelung disease; postinfectious interstitial lung disease; gouty arthritis;autoimmune hepatitis; type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis); type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis); autoimmune mediated hypoglycaemia; type B insulin resistancewith acanthosis nigricans; hypoparathyroidism; osteoarthrosis; primarysclerosing cholangitis; psoriasis type 1; psoriasis type 2; idiopathicleucopaenia; autoimmune neutropaenia; renal disease NOS;glomerulonephritides; microscopic vasculitis of the kidneys; Lymedisease; discoid lupus erythematosus; idiopathic male infertility;nitric oxide-associated male infertility; sperm autoimmunity; multiplesclerosis (all subtypes, including primary progressive, secondaryprogressive, relapsing remitting); sympathetic ophthalmia; pulmonaryhypertension secondary to connective tissue disease; Goodpasture'ssyndrome; pulmonary manifestation of polyarteritis nodosa; acuterheumatic fever; rheumatoid spondylitis; Still's disease; systemicsclerosis; Sjörgren's syndrome; Takayasu's disease/arteritis; autoimmunethrombocytopaenia (AITP); idiopathic thrombocytopaenia; autoimmunethyroid disease; hyperthyroidism; goitrous autoimmune hypothyroidism(Hashimoto's disease); atrophic autoimmune hypothyroidism; primarymyxoedema; phacogenic uveitis; primary vasculitis; vitiligo; acute liverdisease; chronic liver disease; alcoholic cirrhosis; alcohol-inducedliver injury; cholestasis; idiosyncratic liver disease; drug-inducedhepatits; non-alcoholic steatohepatitis; allergy; group B Streptococci(GBS) infection; mental disorders (e.g., depression and schizophrenia);Th2 Type and Th1 Type mediated diseases; acute and chronic pain(different forms of pain); cancer (such as lung, breast, stomach,bladder, colon, pancreas, ovarian, prostate, and rectal cancer);hematopoietic malignancies; leukemia; lymphoma; abetalipoproteinemia;acrocyanosis; acute and chronic parasitic or infectious processes; acuteleukemia; acute lymphoblastic leukemia (ALL); T-cell ALL; FAB ALL; acutemyeloid leukemia (AML); acute or chronic bacterial infection; acutepancreatitis; acute renal failure; adenocarcinomas; atrial ectopicbeats; AIDS dementia complex; alcohol-induced hepatitis; allergicconjunctivitis; allergic contact dermatitis; allergic rhinitis;allograft rejection; alpha-1-antitrypsin deficiency; amyotrophic lateralsclerosis; anemia; angina pectoris; anterior horn cell degeneration;anti-CD3 therapy; antiphospholipid syndrome; anti-receptorhypersensitivity reactions; aortic and peripheral aneurysms; aorticdissection; arterial hypertension; arteriosclerosis; arteriovenousfistula; ataxia; atrial fibrillation (sustained or paroxysmal); atrialflutter; atrioventricular block; B cell lymphoma; bone graft rejection;bone marrow transplant (BMT) rejection; bundle branch block; Burkitt'slymphoma; burns; cardiac arrhythmias; cardiac stun syndrome; cardiactumors; cardiomyopathy; cardiopulmonary bypass inflammation response;cartilage transplant rejection; cerebellar cortical degenerations;cerebellar disorders; chaotic or multifocal atrial tachycardia;chemotherapy associated disorders; chronic myelocytic leukemia (CML);chronic alcoholism; chronic inflammatory pathologies; chroniclymphocytic leukemia (CLL); chronic obstructive pulmonary disease(COPD); chronic salicylate intoxication; colorectal carcinoma;congestive heart failure; conjunctivitis; contact dermatitis; corpulmonale; coronary artery disease; Creutzfeldt-Jakob disease; culturenegative sepsis; cystic fibrosis; cytokine therapy associated disorders;dementia pugilistica; demyelinating diseases; dengue hemorrhagic fever;dermatitis; dermatologic conditions; diabetes mellitus; diabeticarteriosclerotic disease; diffuse Lewy body disease; dilated congestivecardiomyopathy; disorders of the basal ganglia; Down's syndrome inmiddle age; drug-induced movement disorders induced by drugs which blockCNS dopamine receptors; drug sensitivity; eczema; encephalomyelitis;endocarditis; endocrinopathy; epiglottitis; Epstein-Barr virusinfection; erythromelalgia; extrapyramidal and cerebellar disorders;familial hemophagocytic lymphohistiocytosis; fetal thymus implantrejection; Friedreich's ataxia; functional peripheral arterialdisorders; fungal sepsis; gas gangrene; gastric ulcer; glomerularnephritis; graft rejection of any organ or tissue; gram negative sepsis;gram positive sepsis; granulomas due to intracellular organisms; hairycell leukemia; Hallervorden-Spatz disease; Hashimoto's thyroiditis; hayfever; heart transplant rejection; hemochromatosis; hemodialysis;hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura;hemorrhage; hepatitis A; His bundle arrhythmias; HIV infection/HIVneuropathy; Hodgkin's disease; hyperkinetic movement disorders;hypersensitivity reactions; hypersensitivity pneumonitis; hypertension;hypokinetic movement disorders; hypothalamic-pituitary-adrenal axisevaluation; idiopathic Addison's disease; idiopathic pulmonary fibrosis(IPF); antibody mediated cytotoxicity; asthenia; infantile spinalmuscular atrophy; inflammation of the aorta; influenza a; ionizingradiation exposure; iridocyclitis/uveitis/optic neuritis;ischemia-reperfusion injury; ischemic stroke; juvenile rheumatoidarthritis; juvenile spinal muscular atrophy; Kaposi's sarcoma; kidneytransplant rejection; legionella; leishmaniasis; leprosy; lesions of thecorticospinal system; lipedema; liver transplant rejection; lymphedema;malaria; malignant lymphoma; malignant histiocytosis; malignantmelanoma; meningitis; meningococcemia; metabolic syndrome migraineheadache; idiopathic migraine headache; mitochondrial multisystemdisorder; mixed connective tissue disease; monoclonal gammopathy;multiple myeloma; multiple systems degenerations (Menzel;Dejerine-Thomas; Shy-Drager; and Machado-Joseph); myasthenia gravis;mycobacterium avium intracellulare; mycobacterium tuberculosis;myelodysplastic syndrome; myocardial infarction; myocardial ischemicdisorders; nasopharyngeal carcinoma; neonatal chronic lung disease;nephritis; nephrosis; neurodegenerative diseases; neurogenic I muscularatrophies; neutropenic fever; non-Hodgkin's lymphoma; occlusion of theabdominal aorta and its branches; occlusive arterial disorders; OKT3®therapy; orchitis/epididymitis; orchitis/vasectomy reversal procedures;organomegaly; osteoporosis; pancreas transplant rejection; pancreaticcarcinoma; paraneoplastic syndrome/hypercalcemia of malignancy;parathyroid transplant rejection; pelvic inflammatory disease; perennialrhinitis; pericardial disease; peripheral atherosclerotic disease;peripheral vascular disorders; peritonitis; pernicious anemia;pneumocystis carinii pneumonia; pneumonia; POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome); post perfusion syndrome; post pump syndrome;post-MI cardiotomy syndrome; preeclampsia; progressive supranucleopalsy; primary pulmonary hypertension; radiation therapy; Raynaud'sphenomenon; Raynaud's disease; Refsum's disease; regular narrow QRStachycardia; renovascular hypertension; reperfusion injury; restrictivecardiomyopathy; sarcomas; senile chorea; senile dementia of Lewy bodytype; seronegative arthropathies; shock; sickle cell anemia; skinallograft rejection; skin changes syndrome; small bowel transplantrejection; solid tumors; specific arrhythmias; spinal ataxia;spinocerebellar degenerations; streptococcal myositis; structurallesions of the cerebellum; subacute sclerosing panencephalitis; syncope;syphilis of the cardiovascular system; systemic anaphylaxis; systemicinflammatory response syndrome; systemic onset juvenile rheumatoidarthritis; telangiectasia; thromboangitis obliterans; thrombocytopenia;toxicity; transplants; trauma/hemorrhage; type III hypersensitivityreactions; type IV hypersensitivity; unstable angina; uremia; urosepsis;urticaria; valvular heart diseases; varicose veins; vasculitis; venousdiseases; venous thrombosis; ventricular fibrillation; viral and fungalinfections; viral encephalitis/aseptic meningitis; viral-associatedhemophagocytic syndrome; Wernicke-Korsakoff syndrome; Wilson's disease;xenograft rejection of any organ or tissue; acute coronary syndromes;acute idiopathic polyneuritis; acute inflammatory demyelinatingpolyradiculoneuropathy; acute ischemia; adult Still's disease; alopeciagreata; anaphylaxis; anti-phospholipid antibody syndrome; aplasticanemia; arteriosclerosis; atopic eczema; atopic dermatitis; autoimmunedermatitis; autoimmune disorder associated with Streptococcus infection;autoimmune enteropathy; autoimmune hearing loss; autoimmunelymphoproliferative syndrome (ALPS); autoimmune myocarditis; autoimmunepremature ovarian failure; blepharitis; bronchiectasis; bullouspemphigoid; cardiovascular disease; catastrophic antiphospholipidsyndrome; celiac disease; cervical spondylosis; chronic ischemia;cicatricial pemphigoid; clinically isolated syndrome (CIS) with risk formultiple sclerosis; conjunctivitis; childhood onset psychiatricdisorder; dacryocystitis; dermatomyositis; diabetic retinopathy; diskherniation; disk prolapse; drug induced immune hemolytic anemia;endocarditis; endometriosis; endophthalmitis; episcleritis; erythemamultiforme; erythema multiforme major; gestational pemphigoid;Guillain-Barré syndrome (GBS); hay fever; Hughes syndrome; idiopathicParkinson's disease; idiopathic interstitial pneumonia; IgE-mediatedallergy; immune hemolytic anemia; inclusion body myositis; infectiousocular inflammatory disease; inflammatory demyelinating disease;inflammatory heart disease; inflammatory kidney disease; iritis;keratitis; keratojunctivitis sicca; Kussmaul disease or Kussmaul-Meierdisease; Landry's paralysis; Langerhan's cell histiocytosis; livedoreticularis; macular degeneration; microscopic polyangiitis; MorbusBechterev; motor neuron disorders; mucous membrane pemphigoid; multipleorgan failure; myasthenia gravis; myelodysplastic syndrome; myocarditis;nerve root disorders; neuropathy; non-A non-B hepatitis; optic neuritis;osteolysis; pauciarticular JRA; peripheral artery occlusive disease(PAOD); peripheral vascular disease (PVD); peripheral artery; disease(PAD); phlebitis; polyarteritis nodosa (or periarteritis nodosa);polychondritis; polymyalgia rheumatica; poliosis; polyarticular JRA;polyendocrine deficiency syndrome; polymyositis; polymyalgia rheumatica(PMR); post-pump syndrome; primary Parkinsonism; secondary Parkinsonism;prostatitis; pure red cell aplasia; primary adrenal insufficiency;recurrent neuromyelitis optica; restenosis; rheumatic heart disease;SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis);secondary amyloidosis; shock lung; scleritis; sciatica; secondaryadrenal insufficiency; silicone associated connective tissue disease;Sneddon-Wilkinson dermatosis; spondylitis ankylo sans; Stevens-Johnsonsyndrome (SJS); systemic inflammatory response syndrome; temporalarteritis; toxoplasmic retinitis; toxic epidermal necrolysis; transversemyelitis; TRAPS (tumor necrosis factor receptor type 1 (TNFR)-associatedperiodic syndrome); type B insulin resistance with acanthosis nigricans;type 1 allergic reaction; type II diabetes; urticaria; usualinterstitial pneumonia (UIP); vernal conjunctivitis; viral retinitis;Vogt-Koyanagi-Harada syndrome (VKH syndrome); wet macular degeneration;wound healing; Yersinia and Salmonella associated arthropathy.
 50. Themethod according to claim 48, wherein said administering to the subjectis by at least one mode selected from parenteral, subcutaneous,intramuscular, intravenous, intra-articular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.51. A method of treating a patient suffering from a disorder in whichIL-1 is detrimental comprising the step of administering a bindingprotein or a crystal thereof before, concurrently with, or after theadministration of a second agent, wherein the binding protein comprisesfirst and second polypeptide chains, wherein said first polypeptidechain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a firstheavy chain variable domain; VD2 is a second heavy chain variabledomain; C is a heavy chain constant domain; X1 is a linker with theproviso that it is not CH1; X2 is an Fc region; and n is independently 0or 1; and wherein said second polypeptide chain comprises a secondVD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first light chain variabledomain; VD2 is a second light chain variable domain; C is a light chainconstant domain; X1 is a linker with the proviso that it is not CH1; X2does not comprise an Fc region; and n is independently 0 or 1; wherein,in said first polypeptide chain, VD1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 60-148, 196, 198, 200,202, 204, 206, 208 and 210; and VD2 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 213 and 227; wherein,in said second polypeptide chain, VD1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 149-189, 197, 199,201, 203, 205, 207, 209 and 211; and VD2 comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 216 and 229;and wherein the binding protein binds human IL-1β and human IL-1;wherein the second agent is selected from the group consisting of: anantibody, or fragment thereof, capable of binding human IL-1β; anantibody, or fragment thereof, capable of binding human IL-1α;methotrexate; a corticosteroid; a beta-agonist; an antagonist of aleukotriene; an antagonist of ar leukotriene receptor; ADVAIR; an IgEinhibitor; an anti-IgE antibody; XOLAIR; a phosphodiesterase inhibitor;a PDE4 inhibitor; a xanthine; an anticholinergic drug; a mastcell-stabilizing agent; Cromolyn; an IL-4 inhibitor; an IL-5 inhibitor;an eotaxin/CCR3 inhibitor; an antagonist of histamine; an antagonist ofa histamine receptor, an antagonist of prostaglandin D; an inhibitor ofreceptor DP1; an inhibitor of CRTH2; a TNF antagonist; a solublefragment of a TNF receptor; ENBREL; a TNF enzyme antagonist; a TNFconverting enzyme (TACE) inhibitor; a muscarinic receptor antagonist; aTGF-beta antagonist; interferon gamma; perfenidone; a chemotherapeuticagent, leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779;a COX2 or cPLA2 inhibitor; a non-steroidal anti-inflammatory drug(NSAID); an immunomodulator; a p38 inhibitor; a TPL-2 inhibitor; an MK-2inhibitor; an NFκB inhibitor; budenoside; epidermal growth factor;cyclosporine; sulfasalazine; an amino salicylate; 6-mercaptopurine;azathioprine; metronidazole; a lipoxygenase inhibitor; mesalamine;olsalazine; balsalazide; an antioxidant; a thromboxane inhibitor; anIL-1 receptor antagonist; a growth factor; an elastase inhibitor; apyridinyl-imidazole compound; an antibody or agonists of TNF, LT, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24,IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II,GM-CSF, FGF, or PDGF; an antibody to CD2, CD3, CD4, CD8, CD25, CD28,CD30, CD40, CD45, CD69, CD90; FK506; mycophenolate mofetil; ibuprofen;prednisolone; an adensosine agonist; an antithrombotic agent; acomplement inhibitor; an adrenergic agent; a kinase inhibitor; an IL-1βconverting enzyme inhibitors; a T-cell signaling inhibitor; ametalloproteinase inhibitor; an angiotensin converting enzyme inhibitor;soluble cytokine receptor; soluble p55 TNF receptor; soluble p75 TNFreceptor; soluble IL-1RI; soluble IL-1RII; soluble IL-6R; ananti-inflammatory cytokine; IL-4; IL-10; IL-11; and TGF-β. 52-122.(canceled)
 123. A method for treating a subject for a disorder in whichIL-1 activity is detrimental comprising administering to the subject abinding protein or a crystal thereof, wherein the binding proteincomprises first and second polypeptide chains, wherein said firstpolypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein: VD 1is a first heavy chain variable domain; VD2 is a second heavy chainvariable domain; C is a heavy chain constant domain; X1 is a linker withthe proviso that it is not CH1; X2 is an Fc region; and n isindependently 0 or 1; and wherein said second polypeptide chaincomprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first lightchain variable domain; VD2 is a second light chain variable domain; C isa light chain constant domain; X1 is a linker with the proviso that itis not CH1; X2 does not comprise an Fc region; and n is independently 0or 1; wherein, in said first polypeptide chain, VD1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 213 and227; and VD2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 60-148, 196, 198, 200, 202, 204, 206, 208 and210; wherein, in said second polypeptide chain, VD1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 216 and229; and VD2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 149-189, 197, 199, 201, 203, 205, 207, 209 and211; and wherein the binding protein binds human IL-1β and human IL-1α,such that treatment of the disorder is achieved.
 124. The method ofclaim 123, wherein the disorder is selected from the group consistingof: diabetes; uveitis; neuropathic pain; osteoarthritic pain;inflammatory pain; rheumatoid arthritis; osteoarthritis; juvenilechronic arthritis; septic arthritis; Lyme arthritis; psoriaticarthritis; reactive arthritis; spondyloarthropathy; systemic lupuserythematosus (SLE); Crohn's disease; ulcerative colitis; inflammatorybowel disease; autoimmune diabetes; insulin dependent diabetes mellitus;thyroiditis; asthma; allergic diseases; psoriasis; dermatitis;scleroderma; graft versus host disease; organ transplant rejection;acute immune disease associated with organ transplantation; chronicimmune disease associated with organ transplantation; sarcoidosis;atherosclerosis; disseminated intravascular coagulation (DIC);Kawasaki's disease; Grave's disease; nephrotic syndrome; chronic fatiguesyndrome; Wegener's granulomatosis; Henoch-Schoenlein purpurea;microscopic vasculitis of the kidneys; chronic active hepatitis;autoimmune uveitis; septic shock; toxic shock syndrome; sepsis syndrome;cachexia; infectious diseases; parasitic diseases; acute transversemyelitis; Huntington's chorea; Parkinson's disease; Alzheimer's disease;stroke; primary biliary cirrhosis; hemolytic anemia; malignancies; heartfailure; myocardial infarction; Addison's disease; sporadicpolyglandular deficiency type I; polyglandular deficiency type II(Schmidt's syndrome); acute respiratory distress syndrome (ARDS);alopecia; alopecia greata; seronegative arthropathy; arthropathy;Reiter's disease; psoriatic arthropathy; ulcerative colitic arthropathy;enteropathic synovitis; chlamydia; Yersinia and Salmonella associatedarthropathy; spondyloarthropathy; atheromatous disease/arteriosclerosis;atopic allergy; autoimmune bullous disease; pemphigus vulgaris;pemphigus foliaceus; pemphigoid; linear IgA disease; autoimmunehaemolytic anaemia; Coombs positive haemolytic anaemia; acquiredpernicious anaemia; juvenile pernicious anaemia; myalgicencephalitis/Royal Free disease; chronic mucocutaneous candidiasis;giant cell arteritis (GCA); primary sclerosing hepatitis; cryptogenicautoimmune hepatitis; acquired immunodeficiency syndrome (AIDS);acquired immunodeficiency related diseases; hepatitis B; hepatitis C;common varied immunodeficiency (common variable hypogammaglobulinaemia);dilated cardiomyopathy; female infertility; ovarian failure; prematureovarian failure; fibrotic lung disease; cryptogenic fibrosingalveolitis; post-inflammatory interstitial lung disease; interstitialpneumonitis; connective tissue disease associated interstitial lungdisease; mixed connective tissue disease associated lung disease;systemic sclerosis associated interstitial lung disease; rheumatoidarthritis associated interstitial lung disease; systemic lupuserythematosus associated lung disease; dermatomyositis/polymyositisassociated lung disease; Sjögren's disease associated lung disease;ankylosing spondylitis associated lung disease; vasculitic diffuse lungdisease; haemosiderosis associated lung disease; drug-inducedinterstitial lung disease; fibrosis; radiation fibrosis; bronchiolitisobliterans; chronic eosinophilic pneumonia; lymphocytic infiltrativelung disease; postinfectious interstitial lung disease; gouty arthritis;autoimmune hepatitis; type-1 autoimmune hepatitis (classical autoimmuneor lupoid hepatitis); type-2 autoimmune hepatitis (anti-LKM antibodyhepatitis); autoimmune mediated hypoglycaemia; type B insulin resistancewith acanthosis nigricans; hypoparathyroidism; osteoarthrosis; primarysclerosing cholangitis; psoriasis type 1; psoriasis type 2; idiopathicleucopaenia; autoimmune neutropaenia; renal disease NOS;glomerulonephritides; microscopic vasculitis of the kidneys; Lymedisease; discoid lupus erythematosus; idiopathic male infertility;nitric oxide-associated male infertility; sperm autoimmunity; multiplesclerosis (all subtypes, including primary progressive, secondaryprogressive, relapsing remitting); sympathetic ophthalmia; pulmonaryhypertension secondary to connective tissue disease; Goodpasture'ssyndrome; pulmonary manifestation of polyarteritis nodosa; acuterheumatic fever; rheumatoid spondylitis; Still's disease; systemicsclerosis; Sjörgren's syndrome; Takayasu's disease/arteritis; autoimmunethrombocytopaenia (AITP); idiopathic thrombocytopaenia; autoimmunethyroid disease; hyperthyroidism; goitrous autoimmune hypothyroidism(Hashimoto's disease); atrophic autoimmune hypothyroidism; primarymyxoedema; phacogenic uveitis; primary vasculitis; vitiligo; acute liverdisease; chronic liver disease; alcoholic cirrhosis; alcohol-inducedliver injury; cholestasis; idiosyncratic liver disease; drug-inducedhepatits; non-alcoholic steatohepatitis; allergy; group B Streptococci(GBS) infection; mental disorders (e.g., depression and schizophrenia);Th2 Type and Th1 Type mediated diseases; acute and chronic pain(different forms of pain); cancer (such as lung, breast, stomach,bladder, colon, pancreas, ovarian, prostate, and rectal cancer);hematopoietic malignancies; leukemia; lymphoma; abetalipoproteinemia;acrocyanosis; acute and chronic parasitic or infectious processes; acuteleukemia; acute lymphoblastic leukemia (ALL); T-cell ALL; FAB ALL; acutemyeloid leukemia (AML); acute or chronic bacterial infection; acutepancreatitis; acute renal failure; adenocarcinomas; atrial ectopicbeats; AIDS dementia complex; alcohol-induced hepatitis; allergicconjunctivitis; allergic contact dermatitis; allergic rhinitis;allograft rejection; alpha-1-antitrypsin deficiency; amyotrophic lateralsclerosis; anemia; angina pectoris; anterior horn cell degeneration;anti-CD3 therapy; antiphospholipid syndrome; anti-receptorhypersensitivity reactions; aortic and peripheral aneurysms; aorticdissection; arterial hypertension; arteriosclerosis; arteriovenousfistula; ataxia; atrial fibrillation (sustained or paroxysmal); atrialflutter; atrioventricular block; B cell lymphoma; bone graft rejection;bone marrow transplant (BMT) rejection; bundle branch block; Burkitt'slymphoma; burns; cardiac arrhythmias; cardiac stun syndrome; cardiactumors; cardiomyopathy; cardiopulmonary bypass inflammation response;cartilage transplant rejection; cerebellar cortical degenerations;cerebellar disorders; chaotic or multifocal atrial tachycardia;chemotherapy associated disorders; chronic myelocytic leukemia (CML);chronic alcoholism; chronic inflammatory pathologies; chroniclymphocytic leukemia (CLL); chronic obstructive pulmonary disease(COPD); chronic salicylate intoxication; colorectal carcinoma;congestive heart failure; conjunctivitis; contact dermatitis; corpulmonale; coronary artery disease; Creutzfeldt-Jakob disease; culturenegative sepsis; cystic fibrosis; cytokine therapy associated disorders;dementia pugilistica; demyelinating diseases; dengue hemorrhagic fever;dermatitis; dermatologic conditions; diabetes mellitus; diabeticarteriosclerotic disease; diffuse Lewy body disease; dilated congestivecardiomyopathy; disorders of the basal ganglia; Down's syndrome inmiddle age; drug-induced movement disorders induced by drugs which blockCNS dopamine receptors; drug sensitivity; eczema; encephalomyelitis;endocarditis; endocrinopathy; epiglottitis; Epstein-Barr virusinfection; erythromelalgia; extrapyramidal and cerebellar disorders;familial hemophagocytic lymphohistiocytosis; fetal thymus implantrejection; Friedreich's ataxia; functional peripheral arterialdisorders; fungal sepsis; gas gangrene; gastric ulcer; glomerularnephritis; graft rejection of any organ or tissue; gram negative sepsis;gram positive sepsis; granulomas due to intracellular organisms; hairycell leukemia; Hallervorden-Spatz disease; Hashimoto's thyroiditis; hayfever; heart transplant rejection; hemochromatosis; hemodialysis;hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura;hemorrhage; hepatitis A; His bundle arrhythmias; HIV infection/HIVneuropathy; Hodgkin's disease; hyperkinetic movement disorders;hypersensitivity reactions; hypersensitivity pneumonitis; hypertension;hypokinetic movement disorders; hypothalamic-pituitary-adrenal axisevaluation; idiopathic Addison's disease; idiopathic pulmonary fibrosis(IPF); antibody mediated cytotoxicity; asthenia; infantile spinalmuscular atrophy; inflammation of the aorta; influenza a; ionizingradiation exposure; iridocyclitis/uveitis/optic neuritis;ischemia-reperfusion injury; ischemic stroke; juvenile rheumatoidarthritis; juvenile spinal muscular atrophy; Kaposi's sarcoma; kidneytransplant rejection; legionella; leishmaniasis; leprosy; lesions of thecorticospinal system; lipedema; liver transplant rejection; lymphedema;malaria; malignant lymphoma; malignant histiocytosis; malignantmelanoma; meningitis; meningococcemia; metabolic syndrome migraineheadache; idiopathic migraine headache; mitochondrial multisystemdisorder; mixed connective tissue disease; monoclonal gammopathy;multiple myeloma; multiple systems degenerations (Menzel;Dejerine-Thomas; Shy-Drager; and Machado-Joseph); myasthenia gravis;mycobacterium avium intracellulare; mycobacterium tuberculosis;myelodysplastic syndrome; myocardial infarction; myocardial ischemicdisorders; nasopharyngeal carcinoma; neonatal chronic lung disease;nephritis; nephrosis; neurodegenerative diseases; neurogenic I muscularatrophies; neutropenic fever; non-Hodgkin's lymphoma; occlusion of theabdominal aorta and its branches; occlusive arterial disorders; OKT3®therapy; orchitis/epididymitis; orchitis/vasectomy reversal procedures;organomegaly; osteoporosis; pancreas transplant rejection; pancreaticcarcinoma; paraneoplastic syndrome/hypercalcemia of malignancy;parathyroid transplant rejection; pelvic inflammatory disease; perennialrhinitis; pericardial disease; peripheral atherosclerotic disease;peripheral vascular disorders; peritonitis; pernicious anemia;pneumocystis carinii pneumonia; pneumonia; POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome); post perfusion syndrome; post pump syndrome;post-MI cardiotomy syndrome; preeclampsia; progressive supranucleopalsy; primary pulmonary hypertension; radiation therapy; Raynaud'sphenomenon; Raynaud's disease; Refsum's disease; regular narrow QRStachycardia; renovascular hypertension; reperfusion injury; restrictivecardiomyopathy; sarcomas; senile chorea; senile dementia of Lewy bodytype; seronegative arthropathies; shock; sickle cell anemia; skinallograft rejection; skin changes syndrome; small bowel transplantrejection; solid tumors; specific arrhythmias; spinal ataxia;spinocerebellar degenerations; streptococcal myositis; structurallesions of the cerebellum; subacute sclerosing panencephalitis; syncope;syphilis of the cardiovascular system; systemic anaphylaxis; systemicinflammatory response syndrome; systemic onset juvenile rheumatoidarthritis; telangiectasia; thromboangitis obliterans; thrombocytopenia;toxicity; transplants; trauma/hemorrhage; type III hypersensitivityreactions; type IV hypersensitivity; unstable angina; uremia; urosepsis;urticaria; valvular heart diseases; varicose veins; vasculitis; venousdiseases; venous thrombosis; ventricular fibrillation; viral and fungalinfections; viral encephalitis/aseptic meningitis; viral-associatedhemophagocytic syndrome; Wernicke-Korsakoff syndrome; Wilson's disease;xenograft rejection of any organ or tissue; acute coronary syndromes;acute idiopathic polyneuritis; acute inflammatory demyelinatingpolyradiculoneuropathy; acute ischemia; adult Still's disease; alopeciagreata; anaphylaxis; anti-phospholipid antibody syndrome; aplasticanemia; arteriosclerosis; atopic eczema; atopic dermatitis; autoimmunedermatitis; autoimmune disorder associated with Streptococcus infection;autoimmune enteropathy; autoimmune hearing loss; autoimmunelymphoproliferative syndrome (ALPS); autoimmune myocarditis; autoimmunepremature ovarian failure; blepharitis; bronchiectasis; bullouspemphigoid; cardiovascular disease; catastrophic antiphospholipidsyndrome; celiac disease; cervical spondylosis; chronic ischemia;cicatricial pemphigoid; clinically isolated syndrome (CIS) with risk formultiple sclerosis; conjunctivitis; childhood onset psychiatricdisorder; dacryocystitis; dermatomyositis; diabetic retinopathy; diskherniation; disk prolapse; drug induced immune hemolytic anemia;endocarditis; endometriosis; endophthalmitis; episcleritis; erythemamultiforme; erythema multiforme major; gestational pemphigoid;Guillain-Barré syndrome (GBS); hay fever; Hughes syndrome; idiopathicParkinson's disease; idiopathic interstitial pneumonia; IgE-mediatedallergy; immune hemolytic anemia; inclusion body myositis; infectiousocular inflammatory disease; inflammatory demyelinating disease;inflammatory heart disease; inflammatory kidney disease; iritis;keratitis; keratojunctivitis sicca; Kussmaul disease or Kussmaul-Meierdisease; Landry's paralysis; Langerhan's cell histiocytosis; livedoreticularis; macular degeneration; microscopic polyangiitis; MorbusBechterev; motor neuron disorders; mucous membrane pemphigoid; multipleorgan failure; myasthenia gravis; myelodysplastic syndrome; myocarditis;nerve root disorders; neuropathy; non-A non-B hepatitis; optic neuritis;osteolysis; pauciarticular JRA; peripheral artery occlusive disease(PAOD); peripheral vascular disease (PVD); peripheral artery; disease(PAD); phlebitis; polyarteritis nodosa (or periarteritis nodosa);polychondritis; polymyalgia rheumatica; poliosis; polyarticular JRA;polyendocrine deficiency syndrome; polymyositis; polymyalgia rheumatica(PMR); post-pump syndrome; primary Parkinsonism; secondary Parkinsonism;prostatitis; pure red cell aplasia; primary adrenal insufficiency;recurrent neuromyelitis optica; restenosis; rheumatic heart disease;SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis);secondary amyloidosis; shock lung; scleritis; sciatica; secondaryadrenal insufficiency; silicone associated connective tissue disease;Sneddon-Wilkinson dermatosis; spondylitis ankylo sans; Stevens-Johnsonsyndrome (SJS); systemic inflammatory response syndrome; temporalarteritis; toxoplasmic retinitis; toxic epidermal necrolysis; transversemyelitis; TRAPS (tumor necrosis factor receptor type 1 (TNFR)-associatedperiodic syndrome); type B insulin resistance with acanthosis nigricans;type 1 allergic reaction; type II diabetes; urticaria; usualinterstitial pneumonia (UIP); vernal conjunctivitis; viral retinitis;Vogt-Koyanagi-Harada syndrome (VKH syndrome); wet macular degeneration;wound healing; Yersinia and Salmonella associated arthropathy.
 125. Themethod according to claim 123, wherein said administering to the subjectis by at least one mode selected from parenteral, subcutaneous,intramuscular, intravenous, intra-articular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.126. A method of treating a patient suffering from a disorder in whichIL-1 is detrimental comprising the step of administering a bindingprotein or a crystal thereof before, concurrently with, or after theadministration of a second agent, wherein the binding protein comprisesfirst and second polypeptide chains, wherein said first polypeptidechain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a firstheavy chain variable domain; VD2 is a second heavy chain variabledomain; C is a heavy chain constant domain; X1 is a linker with theproviso that it is not CH1; X2 is an Fc region; and n is independently 0or 1; and wherein said second polypeptide chain comprises a secondVD1-(X1)n-VD2-C-(X2)n, wherein: VD1 is a first light chain variabledomain; VD2 is a second light chain variable domain; C is a light chainconstant domain; X1 is a linker with the proviso that it is not CH1; X2does not comprise an Fc region; and n is independently 0 or 1; wherein,in said first polypeptide chain, VD1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 213 and 227; and VD2comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 60-148, 196, 198, 200, 202, 204, 206, 208 and 210; wherein,in said second polypeptide chain, VD1 comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 216 and 229; and VD2comprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 149-189, 197, 199, 201, 203, 205, 207, 209 and 211; andwherein the binding protein binds human IL-1β and human IL-1α, whereinthe second agent is selected from the group consisting of: an antibody,or fragment thereof, capable of binding human IL-1β; an antibody, orfragment thereof, capable of binding human IL-1α; methotrexate; acorticosteroid; a beta-agonist; an antagonist of a leukotriene; anantagonist of ar leukotriene receptor; ADVAIR; an IgE inhibitor; ananti-IgE antibody; XOLAIR; a phosphodiesterase inhibitor; a PDE4inhibitor; a xanthine; an anticholinergic drug; a mast cell-stabilizingagent; Cromolyn; an IL-4 inhibitor; an IL-5 inhibitor; an eotaxin/CCR3inhibitor; an antagonist of histamine; an antagonist of a histaminereceptor, an antagonist of prostaglandin D; an inhibitor of receptorDP1; an inhibitor of CRTH2; a TNF antagonist; a soluble fragment of aTNF receptor; ENBREL; a TNF enzyme antagonist; a TNF converting enzyme(TACE) inhibitor; a muscarinic receptor antagonist; a TGF-betaantagonist; interferon gamma; perfenidone; a chemotherapeutic agent,leflunomide; sirolimus (rapamycin) or an analog thereof, CCI-779; a COX2or cPLA2 inhibitor; a non-steroidal anti-inflammatory drug (NSAID); animmunomodulator; a p38 inhibitor; a TPL-2 inhibitor; an MK-2 inhibitor;an NFκB inhibitor; budenoside; epidermal growth factor; cyclosporine;sulfasalazine; an amino salicylate; 6-mercaptopurine; azathioprine;metronidazole; a lipoxygenase inhibitor; mesalamine; olsalazine;balsalazide; an antioxidant; a thromboxane inhibitor; an IL-1 receptorantagonist; a growth factor; an elastase inhibitor; apyridinyl-imidazole compound; an antibody or agonists of TNF, LT, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24,IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, EMAP-II,GM-CSF, FGF, or PDGF; an antibody to CD2, CD3, CD4, CD8, CD25, CD28,CD30, CD40, CD45, CD69, CD90; FK506; mycophenolate mofetil; ibuprofen;prednisolone; an adensosine agonist; an antithrombotic agent; acomplement inhibitor; an adrenergic agent; a kinase inhibitor; an IL-1βconverting enzyme inhibitors; a T-cell signaling inhibitor; ametalloproteinase inhibitor; an angiotensin converting enzyme inhibitor;soluble cytokine receptor; soluble p55 TNF receptor; soluble p75 TNFreceptor; soluble IL-1RI; soluble IL-1RII; soluble IL-6R; ananti-inflammatory cytokine; IL-4; IL-10; IL-11; and TGF-β.